Cell-free aspirated adipose tissue-derived preparation, composition containing the preparation, and use thereof

Abstract

The present invention relates to cell-free aspirated adipose tissue-derived preparations, compositions containing the same, and their use for therapeutic and / or cosmetic treatment of the skin.

Description

Technical Field

[0001] The present invention belongs to the fields of medicine and cosmetics, particularly cell-free preparations derived from aspirated adipose tissue, compositions containing the same, and their use for therapeutic and / or cosmetic treatment of the skin.

Background Art

[0002] The human skin covers the outside of the body and acts as an important protective barrier against sunlight, heat, pathogens, chemicals, injuries, and other external factors. Sometimes, life damages the skin, makes it aesthetically unpleasant, and functionally impaired. More than 100 million patients obtain surgical scars every year, and millions of people are troubled by chronic skin conditions and traumatic skin defects. Furthermore, the skin is affected by aging and environmental factors. This can lead to lifestyle restrictions, reduced quality of life, and mental distress.

[0003] Skin regeneration is a natural skin regeneration process and is essential for maintaining skin health and vitality as well as for healing traumatic and other skin defects.

[0004] Adipose tissue has been proposed as a source for preparing various products for various medical and cosmetic purposes. Such products include, for example, nanofat, which is obtained by sedimenting aspirated adipose tissue to separate it into three different layers, collecting and emulsifying the intermediate layer containing adipocytes while discarding the other layers. Such products contain cellular components and therefore have limitations in clinical practice. These limitations can potentially be overcome by further processing the nanofat by removing its cellular components. For example, Ziyou Yu et al. (Stem Cell Research & Therapy, 2018, 9(1):1-4), Yunfan He et al. (Stem Cell Research & Therapy, 2019, 10(1):1-4), and Yuda Xu et al. (Aesthetic Surgery Journal, 2020, 40(8):904-913) have all disclosed cell-free products obtained from nanofat.

[0005] U.S. Patent No. 9,631,176 discloses an enzyme-free process for obtaining stem cells from aspirated adipose tissue.

[0006] WO2011 / 019822 and Zhaoyang et al. (Journal of Biomedical Materials Research Part A, 220, 109(8):1418-1428) disclose processed adipose tissue products composed of decellularized adipose tissue extracellular matrix. Such compositions are obtained by chemical or mechanical removal of the cellular compartments of adipose tissue. The products are therefore cell-free and possess the native structure of adipose tissue despite containing little or no lipid residue.

[0007] While several skin regeneration-promoting techniques and products have been developed, their effectiveness remains insufficient. Therefore, there is still a well-recognized need for homogeneous products that can effectively and safely enhance skin regeneration. [Overview of the Initiative]

[0008] In one embodiment, the present invention provides a cell-free aspirated adipose tissue-derived preparation comprising at least one of the lipid fraction and aqueous fraction of aspirated adipose tissue. In other words, the preparation does not contain the adipose tissue fraction of aspirated adipose tissue, which includes adipocytes and other cells naturally present in human adipose tissue.

[0009] In another embodiment, the present invention provides a method for producing the cell-free preparation of the present invention.

[0010] In another aspect, the present invention provides cosmetic and pharmaceutical compositions comprising a cell-free preparation of the present invention and a physiologically acceptable carrier, adjuvant, and / or excipient.

[0011] The invention also provides for the use of cell-free preparations and cosmetic compositions for cosmetic treatment of the skin.

[0012] In a further embodiment, the present invention provides a non-therapeutic method for cosmetically treating the skin, comprising applying the cell-free preparation or cosmetic preparation of the present invention to the skin.

[0013] In a further embodiment, the present invention provides a cell-free preparation or pharmaceutical composition for use in treating clinical skin conditions.

[0014] In a further embodiment, the present invention provides a cell-free preparation or pharmaceutical composition for use in treating conditions associated with fibrosis.

[0015] In a further embodiment, the present invention provides a non-therapeutic method for promoting one or more of the following effects: activation of keratinocytes, activation of fibroblasts, induction of mesenchymal stem cells, and induction of endogenous production of collagen I, collagen III, collagen IV, collagen VIII, elastin, EGF, and / or MMP-1.

[0016] In a further embodiment, the present invention provides the use of the cell-free preparation of the present invention for producing cosmetic compositions for non-therapeutic treatment of the skin.

[0017] Further aspects, embodiments, and details are described in the following drawings, embodiments, examples, and dependent claims for carrying out the invention.

[0018] The attached drawings illustrate several embodiments of the disclosed subject matter and, together with the description, serve to illustrate the principles of the disclosed compositions and methods. [Brief explanation of the drawing]

[0019] [Figure 1] Figure 1 shows right and left profile images of a male whose temples and cheeks, which had acne scars, were treated with laser resurfacing using (right) or without (left) topical administration of the cell-free aspirated adipose tissue preparation of the present invention. The images show the healing and re-epithelialization process two days after the procedure. [Figure 2] Figure 2 shows photographs of a leg that underwent four surgeries with a 3-year-old scar treated with laser surfacing, taken at various time points (6 hours, 2 days, and 28 days) after the procedure. The lower half of the scar was treated twice with topical administration of the cell-free aspirated adipose tissue preparation of the present invention. [Figure 3] Figure 3 shows photographs of a skin area with some degree of scarring before (left) and 4 weeks after (right) topical administration of the cell-free aspirated adipose tissue preparation of the present invention, following microneedling treatment. [Figure 4] Figure 4 shows photographs of the eye and mouth area before (left) and 4 weeks after (right) mesotherapy using the cell-free aspirated adipose tissue preparation of the present invention. [Figure 5] Figure 5 shows fibroblast migration as a function of time. The dotted line (A) shows migration in the medium control group, while the solid line (B) shows migration in the group exposed to cell-free aspirated adipose tissue preparation. Significant differences at each time point are indicated by asterisks, where a single asterisk (*) indicates p-values ​​from <0.01 to 0.001, and two asterisks (**) indicate p-values ​​from <0.01 to 0.001. [Figure 6] Figures 6A and 6B show differential gene expression after 6 days of exposure of BJ fibroblasts to a cell-free aspirated adipose tissue preparation (B) compared to a medium control (A), as a ratio of change for the importin 8 (IPO8) housekeeping gene. Figure 6A shows the expression of smooth muscle actin (ACTA2), collagen 1 (COL1), collagen 3 (COL3), collagen 8 (COL8), and elastin (ELN), while Figure 6B shows the expression of collagen 4 (COL4A). Asterisks indicate the significance of differential expression, with one asterisk (*) indicating p-values ​​from <0.05 to 0.01, two asterisks (**) indicating p-values ​​from <0.01 to 0.001, and three asterisks (*) indicating p-values ​​less than 0.001. [Figure 7] Figure 7 shows triglyceride formation in human adipose stem cells after exposure to a media control (A) and two different concentrations of cell-free aspirated adipose tissue-derived preparations, 0.8 mg / ml (B) and 0.4 mg / ml (C). The significance of the changes was calculated relative to the media control. Significance is indicated by an asterisk, where a single asterisk (*) represents p-values ​​from <0.05 to 0.01, and two asterisks (**) represent p-values ​​from <0.01 to 0.001. [Figure 8]Figure 8 shows the differential gene expression after 6 days of exposure of human adipose stem cells to cell-free aspirated adipose tissue-derived preparation (B) compared to media control (A). Figure 8 shows the expression of smooth muscle actin (ACTA2) and collagen 1 (COL1). Significance is indicated as an asterisk, where p-values less than 0.05 to 0.01 are indicated by one asterisk (*). [Figure 9] Figure 9 shows increased keratinocyte proliferation after 6 days of exposure to cell-free aspirated adipose tissue-derived preparation (B) normalized to control (A), with a value of 1 given to the control. The significant difference is indicated by an asterisk, where p-values less than 0.01 to 0.001 are indicated by one asterisk (*). [Figure 10] Figure 10 shows keratinocyte migration as a function of time (days). The dotted line (A) shows the migration of the media control, while the solid line (B) shows the migration after exposure to the cell-free aspirated adipose tissue-derived preparation. The significant difference at each time point is indicated by an asterisk, where p-values less than 0.01 to 0.001 are indicated by one asterisk (*) and p-values less than 0.01 to 0.001 are indicated by two asterisks (**). [Figure 11] Figures 11A, 11B, and 11C show the differential gene expression after 15 days of exposure of keratinocytes to cell-free aspirated adipose tissue-derived preparation (B) compared to media control (A). Figure 11A shows the expression of epidermal growth factor (EGF), Figure 11B shows the expression of matrix metalloproteinase 1 (MMP1), and Figure 11C shows the expression of TIMP metallopeptidase inhibitor 1, tissue inhibitor of metalloproteinase 1 (TIMP-1). Significance is indicated as an asterisk, where p-values less than 0.05 to 0.01 are indicated by one asterisk (*). [Figure 12]Figure 12 shows selected enrichment terms from the sub - ontologies of Gene Ontology (GO), namely Biological Process (BP), Cellular Component (CC), and Molecular Function (MF). The x - axis shows the common logarithm of the p - values calculated in the enrichment analysis, and the dot size shows the number of genes in each term. The GO terms on the y - axis are: 1 = angiogenesis, 2 = regulation of peptidase activity, 3 = tissue migration, 4 = immune system process, 5 = fatty acid metabolic process, 6 = response to oxidative stress, 7 = blood coagulation, 8 = regulation of organization of cellular components, 9 = wound healing, 10 = cellular detoxification, 11 = organization of supramolecular fibers, 12 = proteolysis, 13 = immunoglobulin complex, 14 = high - density lipoprotein particle, 15 = extracellular matrix, 16 = collagen - containing extracellular matrix, 17 = extracellular exosome, 18 = lipid binding, 19 = glycosaminoglycan binding, 20 = structural constituent of extracellular matrix, 21 = antioxidant activity, 22 = activity of peptidase regulator, 23 = activity of oxidoreductase, 24 = binding of cytoskeletal protein, 25 = binding of cell adhesion molecule.

Mode for Carrying Out the Invention

[0020] The following detailed description illustrates aspects and embodiments of the present invention and the ways in which they can be implemented. Although some modes for carrying out the present invention are disclosed, it is understood that other embodiments for implementing or executing the present invention are also possible. In other words, the scope of the present invention is limited only by the appended claims.

[0021] It is also understood that, unless otherwise specified, the terms used in this specification have the same meaning as those commonly understood by a person skilled in the art to which the present invention pertains.

[0022] The singular nouns used in this specification also carry the meaning of the corresponding plural nouns, unless otherwise specified. In other words, unless otherwise specified, the singular expressions "a", "an", and "the" have the meaning of "one" as well as "one or more".

[0023] The term "and / or" in phrases such as "X and / or Y" should be interpreted as meaning either "X and Y" or "X or Y," and should be interpreted as providing explicit support for both meanings or either meaning.

[0024] The terms “comprising,” “including,” “containing,” and “having” are interchangeable and intended to be interpreted in a non-exclusive manner, in other words, they allow for the existence of matters, components, or elements that are not explicitly stated.

[0025] In one embodiment, the present invention provides a cell-free preparation comprising or consisting of a lipid fraction and / or aqueous fraction of aspirated adipose tissue. Accordingly, the terms “cell-free preparation” and “cell-free aspirated adipose tissue-derived preparation” as used herein are interchangeable and are sometimes abbreviated as “preparation.”

[0026] As used herein, the term "cell-free" refers to a preparation that lacks or is essentially lacking cells. Techniques for determining whether a given preparation is cell-free are readily available in the art.

[0027] As used herein, the term "aspiration adipose tissue" refers to the material obtained by liposuction. This may originate from a single human subject or from a mixture of aspiration adipose tissues from multiple human subjects. Both options are included in the term "aspiration adipose tissue."

[0028] Liposuction is the surgical removal of subcutaneous adipose tissue (i.e., adipose tissue in the subcutaneous tissue) assisted by suction using a suction cannula introduced through a small skin incision. Various liposuction techniques are readily available and are evolving with technological advancements. Some non-exclusive examples of technological advancements in addition to conventional dry liposuction techniques include ultrasonic liposuction, water-assisted liposuction, and radiofrequency-assisted liposuction. A new, cutting-edge liposuction technique is water-assisted liposuction, which utilizes a pressurized flow of saline / aqueous buffer solution approximately 2 to 3 times the volume of subcutaneous fat to be removed. This causes the layer of fatty subcutaneous tissue to swell and harden before suction, facilitating the removal of subcutaneous fat / tissue from various areas of the body. The saline / aqueous buffer solution commonly used in water-assisted liposuction is called a "swelling solution." Therefore, the liposuction technique may be called swelling liposuction. Conventional swelling solutions contain local anesthetics such as lidocaine and adrenaline or epinephrine. It should be noted that the choice of technique often depends on the patient's characteristics and the surgeon's preference; therefore, the present invention is not limited to any particular liposuction technique.

[0029] As used herein, the term “liposuction solution” means any physiologically acceptable aqueous solution suitable for use as an infiltration in any liposuction technique, including conventional edema liposuction as well as other water-assisted liposuction techniques. Non-limiting examples of suitable liposuction solutions include 0.9% sodium chloride (saline), ringer’s acetate, ringer’s fundin, lactated ringer’s solution, and normofundin. Those skilled in the art can easily select an appropriate aqueous buffer solution for each case. In some embodiments, the liposuction solution may contain any approved local anesthetic, such as lidocaine or prilocaine, typically 0.5% or 1% lidocaine (up to 55 mg / kg). In some other embodiments, the liposuction solution may contain adrenaline / epinephrine, typically 1% adrenaline (up to 7 mg / kg), for temporary vasoconstriction to prevent bleeding during liposuction. In some embodiments, the liposuction solution may contain both lidocaine (and / or any other suitable local anesthetic) and adrenaline, preferably in the amounts described above. Such a liposuction solution is conventionally called a swelling solution, but any buffered aqueous solution used in liposuction is sometimes called a “swelling solution,” whether or not lidocaine or adrenaline / epinephrine is present. Therefore, the terms “liposuction solution” and “swelling solution” as used herein are interchangeable unless otherwise specified. Thus, in some further embodiments, a liposuction solution that does not contain either lidocaine (or any other local anesthetic) or adrenaline / epinephrine may still be called a “swelling solution.”

[0030] The term "spent liposuction solution" refers to a liposuction solution used in liposuction, i.e., a liposuction solution that has come into contact with human subcutaneous fat in vivo and / or ex vivo after aspiration. Spent liposuction solution contains a variety of at least partially water-soluble substances derived from subcutaneous adipose tissue.

[0031] When aspirated adipose tissue obtained by a liposuction technique, including the use of a liposuction solution, is centrifuged or allowed to stand for a period of time, the aspirated adipose tissue separates into various layers: an upper lipid layer, an intermediate adipose tissue layer containing adipocytes and other cells of the adipose tissue, and a lower aqueous layer. The aqueous layer contains the used liposuction solution used in the liposuction procedure. Any cells or tissue fragments contained in the aspirated adipose tissue may separate into a fourth layer below the aqueous layer. In this context, the term "layer" can be used interchangeably with the term "fraction."

[0032] As used herein, the term “fraction” is not limited to the entire fraction (i.e., layer) but also includes any portion thereof. In other words, the cell-free preparations of the present invention do not need to contain the entire lipid fraction and / or the entire aqueous fraction of the aspirated adipose tissue. In other words, cell-free preparations containing only a portion and / or only a portion of the lipid fraction of the aspirated adipose tissue are encompassed by the cell-free aspirated adipose tissue-derived preparations described in the claims, which contain at least one of the lipid fraction and aqueous fraction of the aspirated adipose tissue.

[0033] The cell-free preparation of the present invention can be obtained by processing aspirated adipose tissue in a manner that results in a preparation containing lipid fractions and / or aqueous fractions of aspirated adipose tissue and lacking cells naturally present in the adipose tissue. This can be achieved by various methods. In some embodiments, this method includes the steps of removing the adipose tissue fraction from the aspirated adipose tissue, collecting the remaining lipid fraction and / or aqueous fraction, and sterile filtering the collected fraction. In some other embodiments, the adipose tissue fraction is not actively removed, and only the desired fraction, i.e., only the lipid fraction, only the aqueous fraction, or both, is collected and subsequently sterile filtered. Since no washing step is performed before the step of collecting the aqueous fraction, this contains used liposuction solution. Therefore, the collected fraction may or may not contain local anesthetics such as lidocaine and / or adrenaline. Also, the lipid fraction is not removed by any washing step. In some embodiments, the aspirated adipose tissue is gently shaken and / or allowed to settle to separate into various layers before the steps of removing the adipose tissue fraction and / or collecting the lipid / or aqueous fraction. The adipose tissue fraction can be removed by any suitable technique available in the art. The collection of the lipid and / or aqueous fractions can also be carried out by any suitable technique available in the art. Sterile filtration is usually carried out using a 0.22 μm filter, i.e., a membrane filter with a pore size of 0.22 μm. Alternatively, sterile filtration may be carried out using a 0.2 μm filter, i.e., a membrane filter with a pore size of 0.2 μm.

[0034] In some embodiments, liposuction solutions used in liposuction but not aspirated with adipose tissue (i.e., collected in a separate container during or immediately after liposuction) can be provided as cell-free preparations of the present invention, for example by adding them to the collected aqueous fraction of aspirated adipose tissue to increase its volume, or can be used as aqueous components thereof. In some further embodiments, any physiologically acceptable aqueous buffer solution / saline solution may be added to the aspirated adipose tissue to increase the volume of the aqueous fraction, if desired.

[0035] In some embodiments, the used liposuction solution, either as a collected aqueous fraction of aspirated adipose tissue or as a used liposuction solution that does not form part of the aspirated adipose tissue, may be diluted or concentrated as desired by adjusting its volume using physiologically acceptable diluents and / or techniques readily available in the art. In some embodiments, the used liposuction solution may be lyophilized and reconstituted on demand.

[0036] Accordingly, the term "aqueous fraction of aspirated adipose tissue" refers to the lower fraction of aspirated adipose tissue or the lower fraction of used liposuction solution, and the lower fraction is formed when the aspirated adipose tissue or the used liposuction solution is allowed to settle, that is, when it is separated into the layers described above.

[0037] In some embodiments, the preparation of the present invention consists of a lipid fraction of aspirated adipose tissue. In some embodiments, the preparation contains the lipid fraction of aspirated adipose tissue as the sole material derived from aspirated adipose tissue. In some further embodiments, the preparation is provided as a filtered, preferably sterile-filtered, lipid fraction of aspirated adipose tissue. In some even further embodiments, the lipid fraction is sterile-filtered through a 0.2 μm or 0.22 μm filter. In some even further embodiments, the lipid fraction may be provided as a lyophilized preparation that is reconstituted on demand.

[0038] In some embodiments, the preparation of the present invention comprises an aqueous fraction of aspirated adipose tissue and / or a used liposuction solution that is not part of the aspirated adipose tissue. In some embodiments, the preparation of the present invention includes an aqueous fraction of aspirated adipose tissue and / or a used liposuction solution that was not collected as part of the aspirated adipose tissue as the sole liposuction-derived material in the preparation. In some further embodiments, the preparation is a filtered, preferably sterile-filtered, lipid fraction of aspirated adipose tissue and / or a used liposuction solution that is not part of the aspirated adipose tissue. In some even further embodiments, the preparation is sterile-filtered through a 0.2 or 0.22 μm filter. In some even further embodiments, the preparation is provided as a lyophilized preparation that is reconstituted on demand.

[0039] In some embodiments, the preparation of the present invention comprises the lipid and aqueous fractions of aspirated adipose tissue, with or without supplementation of spent liposuction solution that is not part of the aspirated adipose tissue. In some embodiments, the preparation of the present invention comprises the lipid and aqueous fractions of aspirated adipose tissue as the sole liposuction-derived material in the preparation, with or without supplementation of spent liposuction solution that was not collected as part of the aspirated adipose tissue. The lipid fraction may be the total lipid fraction of the aspirated adipose tissue or a portion thereof. In some further embodiments, the preparation comprises the filtered, preferably sterile-filtered, lipid and aqueous fractions of aspirated adipose tissue. In some even further embodiments, the preparation is sterile-filtered through a 0.2 μm or 0.22 μm filter. In some even further embodiments, the preparation may be provided as a lyophilized preparation that is reconstituted on demand.

[0040] In some embodiments, the preparation of the present invention comprises or consists of a used liposuction solution that does not contain adipose tissue fraction, i.e., does not contain intermediate fractions formed when aspirated adipose tissue or used liposuction solution is settled. In some embodiments, the preparation of the present invention comprises a used liposuction solution that does not contain adipose tissue fraction, i.e., does not contain intermediate fractions formed when aspirated adipose tissue or used liposuction solution is settled, as the sole liposuction-derived material in the preparation. In some further embodiments, the preparation is provided as a filtered, preferably sterile-filtered, preparation. In some even further embodiments, the preparation is sterile-filtered through a 0.2 μm or 0.22 μm filter. In some even further embodiments, the preparation may be provided as a lyophilized preparation that is reconstituted on demand.

[0041] In any of the embodiments described above, the preparation may be supplemented with used liposuction solution, which is not part of the actual aspirated adipose tissue.

[0042] In accordance with the foregoing, the preparation of the present invention is - A step of providing aspirated adipose tissue obtained from at least one human subject; - A step of optionally shaking human aspirated adipose tissue; - A step of removing the adipose tissue fraction of aspirated adipose tissue, including cells; - A process of filtering the remaining fraction of aspirated fatty tissue and It can be obtained by a method that includes [a specific method].

[0043] In some further embodiments of the above method, the aspirated adipose tissue is allowed to settle before removing the adipose tissue fraction, thereby separating the aspirated adipose tissue into various layers, namely, an upper lipid layer, an intermediate adipose tissue layer containing adipocytes and other cells of the adipose tissue, and a lower aqueous layer.

[0044] In some other embodiments, the preparations of the present invention are - A step of providing aspirated adipose tissue obtained from at least one human subject, - A step of optionally shaking human aspirated adipose tissue; - A step of discarding the adipose tissue fraction containing cells, while collecting at least one of the lipid fraction and the aqueous fraction; - A process of filtering the collected fractions and It can be obtained by a method that includes [a specific method].

[0045] In some further embodiments of the above method, the aspirated adipose tissue is allowed to settle before the step of collecting at least one of the lipid fraction and the aqueous fraction, thereby separating the aspirated adipose tissue into various layers, namely, an upper lipid layer, an intermediate adipose tissue layer containing adipocytes and other cells of the adipose tissue, and a lower aqueous layer. The lipid and / or aqueous fractions can be collected at least partially.

[0046] In this method, filtration of the remaining or collected fraction limits the particle size of various components in the resulting cell-free preparation, depending on the pore size of the filter used. When a 0.2 μm or 0.22 μm filter is used, the preparation of the present invention does not contain large intact ECM proteins having at least one dimension greater than 0.2 μm or 0.22 μm, respectively. Filtrating the remaining or collected fraction with such a filter also sterilizes the preparation. Therefore, preferred filters are 0.2 μm or 0.22 μm filters.

[0047] Notably, obtaining aspirated adipose tissue from at least one human subject is not part of the method described above. In other words, the method is applied to aspirated adipose tissue that has been previously obtained from the aforementioned at least one human subject.

[0048] One aspect of this disclosure relates to the method described above itself.

[0049] Being cell-free means that this preparation is fundamentally different from any stromal vascular fraction (SVF) derived from adipose tissue that contains heterogeneous cell populations such as mesenchymal precursor / stem cells, preadipocytes, endothelial cells, pericytes, T cells, and M2 macrophages. SVF can be obtained from the adipose tissue fraction of aspirated adipose tissue. Therefore, this preparation is fundamentally different from nanofat, which is the emulsified and filtered adipose tissue fraction of aspirated adipose tissue, as well as any cell-free extract obtained by cell removal from nanofat. In other words, nanofat is obtained by discarding the lipid and aqueous fractions of aspirated adipose tissue and emulsifying the remaining adipose tissue fraction, i.e., the cellular fraction. Even if the emulsified cellular fraction is further processed by removal of cellular components (this process may involve the use of aqueous solutions), it is understood that the resulting cell-free extract will not be recognized as the aqueous fraction of aspirated adipose tissue, even if the cell-free extract derived from nanofat is aqueous.

[0050] Furthermore, the cell-free preparations of the present invention are fundamentally different from any decellularized adipose tissue-derived compositions available in the art. Such decellularized compositions are natural scaffolds derived from adipose tissue, from which cellular and nuclear contents have been removed, but the three-dimensional structure and composition of the extracellular matrix (ECM) are preserved.

[0051] The preparations of the present invention have several advantages due to being cell-free, including, for example, low immunogenicity and the resulting reduced risk of adverse effects when administered to the human body. Therefore, the preparations of the present invention can be provided as products of the same origin, i.e., as products suitable for use by subjects different from aspirated adipose tissue donors. Furthermore, there are numerous practical advantages when factors such as cell viability during storage or after administration do not need to be considered.

[0052] Previous studies have shown that cell-free extracts derived from adipose tissue can be used in soft tissue engineering and repair due to their ability to induce both angiogenesis, i.e., the formation of new blood vessels, and adipogenesis, i.e., the formation of new adipose tissue.

[0053] Unexpectedly, it was discovered that the cell-free adipose tissue-derived preparation of the present invention can promote skin regeneration, a process that does not involve lipogenesis and is not only affected by angiogenesis but also heavily dependent on other cellular processes such as the regeneration of epidermal and dermal cells, particularly the activation of keratinocytes and fibroblasts, respectively.

[0054] Therefore, in some embodiments, cell-free preparations of the present invention comprising or consisting of a lipid fraction and / or aqueous fraction of aspirated adipose tissue may be presented as cell-free allogeneic skin regeneration substitutes.

[0055] The skin is the largest organ in the human body and consists of three layers. The outermost layer of the skin is the epidermis, a thin protective layer mainly composed of keratinocytes. The middle layer of the skin is the dermis, which makes up 90% of the skin's thickness. The dermis is rich in collagen, a protein that makes skin cells strong and elastic, and elastin, a protein that keeps the skin flexible and helps stretched skin regain its shape. The basement membrane region between the epidermis and dermis connects and functionally separates the epidermis and dermis and is essential for normal skin function. The bottom layer of the skin is the subcutaneous tissue (hypodermis) (i.e., subcutaneous tissue (subcutis)), a fatty layer that cushions the underlying muscles and bones. The fat of the subcutaneous tissue is arranged in the form of lobules, separated from each other by fibrous septa consisting of blood vessels, nerves, lymphatic vessels, and connective tissue. Each lobule contains adipocytes (i.e., fat cells) that are mostly composed of triglycerides.

[0056] As used herein, the term "hypodermal" refers to a substance that may be derived from human hypodermal tissue, i.e., subcutaneous tissue. This term may be used interchangeably with the terms "hypodermis-derived" or "subcutis-derived."

[0057] Since the preparation of the present invention is derived from aspirated adipose tissue, it can also be referred to, for example, as a cell-free subcutaneous tissue preparation.

[0058] As used herein, the term "skin regeneration" refers to the natural process that occurs as skin cells undergo metabolic turnover. In other words, dead skin cells in the uppermost layer of the epidermis fall off, revealing fresh, newly created cells directly beneath. If the skin heals but does not regenerate, scar tissue is formed. As we age, skin regeneration slows and alters the basement membrane region, the skin becomes less elastic, thinner, more wrinkled, and has a rougher texture.

[0059] Without being limited to any theory, the preparations of the present invention may promote skin regeneration through several different mechanisms of action, thereby improving the management of scars and other skin defects and reducing the visible signs of skin aging. For example, the preparations not only deliver essential structural components (i.e., constituent units) and nutrients to the skin, but also induce local cells to produce more essential factors. More specifically, as shown in the examples, the preparations activate fibroblasts, which are cells that produce collagen, hyaluronic acid, and elastin, and also induce the migration and differentiation of local cells. The examples also show that the preparations activate keratinocytes and increase their metabolic turnover, thereby improving the skin barrier function. Furthermore, it is envisioned that the preparations improve the functionality and repair of the dermal-epidermal junction.

[0060] Aging skin becomes drier, thinner, loses its firmness, and appears more wrinkled. The outermost layer of skin has a reduced water content, partly due to a decrease in lipids, i.e., fatty substances, and partly due to a decrease in hyaluronic acid. Both of these changes affect the skin, making it no longer able to bind and retain water as it did younger skin. The cell-free preparations of the present invention contain a wide number of diverse lipids, fatty acids, related proteins, and proteins associated with lipid metabolism, which are considered to help the skin retain moisture, thereby reducing its dryness, and thus not only rejuvenate aging skin but also provide a means for managing clinical and other skin conditions and defects that would benefit from reduced skin dryness.

[0061] More specifically, based on non-target compositional analysis of the preparation, it essentially contains lipids, related proteins, and proteins related to lipid metabolism, including, but not limited to, ceramides, apolipoproteins, perilipins, unsaturated and saturated fatty acids, glycerophospholipids, lysophosphatidic acid, lysophospholipids, monoglycerides, diglycerides, triglycerides, and prostaglandins. Some of these molecules may originate from the lipid fraction of aspirated adipose tissue, while some other molecules may originate from the aqueous fraction of aspirated adipose tissue. Thus, some components in the preparation may be lipid-soluble, while some other components may be water-soluble or partially water-soluble. Ceramics are particularly interesting components of the preparation according to the present invention because they are lipids found in normal skin cells.

[0062] Characterization of the preparations of the present invention, according to other proposed mechanisms of action, reveals that they also essentially contain structural and non-structural extracellular matrix proteins, peptides, chains, subchains, and / or their subunits, as well as proteins involved in the synthesis of the extracellular matrix. These include, but are not limited to, collagen I, III, IV, VI, XV, XVIII, fibronectin, vitronectin, elastin, hyaluronan, decorin, tenascin, laminin, lumican, and prolargin. Interestingly, lumican is a leucine-rich repeat proteoglycan that induces collagen fibrillation, while prolargin is a protein that anchors the basement membrane to the underlying tissue.

[0063] Interestingly, the preparations of the present invention contain basement membrane proteins, peptides, chains, subchains, and / or their subunits, specialized dermal-epidermal junction components, and factors affecting the epidermis, including, for example, but not limited to, keratin, nidogen, versican, integrin, periplakin, and plectin.

[0064] This preparation may contain components of the extracellular membrane (ECM) and basement membrane (BM), but it is understood that it does not contain intact ECM or intact BM, and is therefore fundamentally different from any decellularized composition. In this context, intact ECM and / or BM refers to a composition in which the three-dimensional structure and composition of ECM and / or BM are preserved, respectively. The preparation also does not contain any distinct large components of ECM and / or BM. For example, the preparation does not contain intact collagen fibrils, but may contain various collagen molecules, capillaries, or secondary chains. Collagen exists in various forms, including at least collagen I, III, IV, and VIII, and at various molecular sizes. The method for producing the preparation of the present invention does not involve a step of extracting collagen or other large ECM / BM components, nor does it even allow for the presence of such intact components in the resulting preparation.

[0065] Furthermore, in these embodiments, which include a step of filtering through a 0.2 μm or 0.22 μm filter, no components larger than the pore size can be present. It should be noted that intact collagen I fibrils may be significantly larger in size than, for example, the pore size of a 0.2 μm or 0.22 μm filter. Therefore, the filtered preparation may contain various peptides, side chains, and capillary fibers of collagen and other ECM / BM components, but the size of their molecules is limited to 200 nm or 220 nm.

[0066] Notably, the preparations of the present invention also contain essential and non-essential amino acids, antioxidants, oxidoreductases, vitamin derivatives and metabolites, diverse glycoproteins and proteoglycans, and factors related to cell proliferation. Furthermore, while these findings follow the proposed mechanism of action, the present invention is not limited to any theory or mechanism of action. Tretinoin is a particularly interesting component of this preparation, as it is a well-known antioxidant for its anti-aging benefits and is therefore often added to various skincare products. Tretinoin can also be classified as a vitamin, or more specifically, a vitamin derivative or metabolite.

[0067] Surprisingly, among the more than 1004 proteins, more than 6000 related peptides, and more than 500 metabolites identified as components of this preparation based on non-targeted analysis by liquid chromatography-mass spectrometry (LC-MS), neither VEGF, IGF-1, nor FGF-2 were present.

[0068] The cell-free preparation of the present invention can be used for various therapeutic and / or cosmetic treatments of the skin. It can be used on its own or formulated into a pharmaceutical or cosmetic composition.

[0069] As used herein, the term “pharmaceutical composition” broadly refers to a composition comprising the cell-free aspirated adipose tissue preparation of the present invention as a therapeutic active ingredient, and one or more pharmaceutically acceptable components, such as carriers, adjuvants, and / or excipients. As used herein, the term “pharmaceutically acceptable” means a material that is suitable for administration to human subjects without excessive adverse side effects, such as toxicity, significant irritation, and / or allergic reactions. In other words, the benefit-risk ratio must be reasonable. Essentially, the term “pharmaceutically acceptable” is interchangeable with the term “physiologically acceptable.” Furthermore, pharmaceutically acceptable components should not diminish the therapeutic activity of the active ingredient, i.e., the aspirated adipose tissue preparation.

[0070] As used herein, the term “cosmetic composition” broadly refers to a composition comprising the cell-free aspirated adipose tissue preparation of the present invention as an active ingredient, and one or more cosmetically acceptable components, such as carriers, adjuvants, and / or excipients. As used herein, the term “cosmetically acceptable” means a material that is suitable for administration to human subjects without excessive adverse side effects, such as toxicity, significant irritation, and / or allergic reactions. In other words, the benefit-risk ratio must be reasonable. As will be readily apparent to those skilled in the art, for a composition to be “cosmetically acceptable,” it must also be “physiologically acceptable” and “dermatologically acceptable.” These terms can therefore be used interchangeably.

[0071] A person skilled in the art to which this invention belongs can easily select appropriate pharmaceutically and / or cosmetically acceptable components available in the art, depending on the intended route of administration and formulation of the composition. Formulation may be carried out using means and methods readily available in the art, as desired, for example, by conventional mixing, dissolution, granulation, grinding, emulsification, encapsulation, encapsulation, lyophilization, etc.

[0072] Excipients are preferably inert substances added to pharmaceutical and cosmetic compositions. Typical examples of various types of excipients include, but are not limited to, stabilizers, preservatives, pH adjusters, fillers, thickeners, viscosity modifiers, lubricants, solubilizers, and surfactants.

[0073] Examples of pharmaceutical and cosmetic compositions for topical administration include, but are not limited to, ointments, lotions, creams, gels, hydrogels, oil-in-water emulsions, water-in-oil emulsions, droplets, sprays, liquids, solutions, powders, sustained-release or long-lasting-release formulations, face masks, skin patches, mousses, and foams.

[0074] Pharmaceutical and cosmetic compositions for parenteral administration are typically sterile aqueous or non-aqueous solutions, suspensions, or emulsions applied topically or by injection, for example, subcutaneously, intracutaneously, or intradermally. In some embodiments, the composition may be administered into different layers of the skin during the same treatment session and / or process. For example, the composition may be administered both subcutaneously, intracutaneously, and / or intradermally by injection, as well as by topical administration.

[0075] Whether the composition is a pharmaceutical composition or a cosmetic composition, and whether the composition is intended for topical or parenteral administration, the composition may also be provided in a concentrated form or in a powder (i.e., lyophilized) form that can be reconstituted on demand.

[0076] The present invention provides cell-free aspirated adipose tissue-derived preparations disclosed herein for use in aesthetic dermatology. This aspect of the present invention may be expressed, for example, as the use of the cell-free preparation or a cosmetic composition containing the same for the cosmetic treatment of skin, or as a non-therapeutic method for the cosmetic treatment of skin, more particularly selected areas of human skin. The method comprises the step of applying the preparation or the composition to the skin to be treated, preferably in a cosmetically effective amount. In some embodiments, the skin to be treated is damaged skin.

[0077] As used herein, the terms “cosmetic treatment” and “non-therapeutic treatment” may be used interchangeably and both refer to the administration of a cell-free preparation of the present invention or a cosmetic composition containing the same to a subject in need for the purpose of promoting skin regeneration and / or skin rejuvenation. Improved skin regeneration and / or rejuvenation may, but are not limited to, a reduction in dark circles around the eyes, a reduction in skin dryness and roughness, a reduction in wrinkles and fine lines, a reduction in skin redness, a reduction in the number, size, and / or darkness of age spots (melasma) and other hyperpigmentation defects, a reduction in the number, size, and / or whiteness of vitiligo patches, a reduction in cellulite and burns, and improved striae and scars, such as scars associated with acne or other clinical skin conditions, or scars caused by injury or surgery.

[0078] As used herein, the term “cosmetically effective amount” means an amount that at least reduces or improves signs of cosmetic defects, such as dark areas around the eyes, dryness, and rough skin, wrinkles and fine lines, redness of the skin, age spots (melasma), and other hyperpigmentation defects, vitiligo patches, cellulite, striae, and scars.

[0079] As used herein, the term “damaged skin” refers to skin whose outer layer, i.e., the epidermis, is damaged for any reason. In relation to aesthetic dermatology, skin treated may include skin resulting from cosmetic procedures such as microneedling, laser treatment, or exfoliation, or from aging.

[0080] In a further embodiment, the present invention provides a cell-free preparation disclosed herein for use in clinical dermatology. This embodiment of the present invention may be expressed, for example, as a cell-free preparation or a pharmaceutical composition containing the same for use in treating a clinical skin disorder or condition, or as a therapeutic method for treating a clinical skin disorder or condition typically exhibiting damaged skin. The method comprises the step of applying the preparation or composition to the skin to be treated, preferably in a therapeutically effective amount.

[0081] As used herein, the term “therapeutic treatment” means, in its various linguistic forms, the administration of a cell-free preparation of the present invention or a pharmaceutical composition containing it to a subject in need, for the purpose of improving, alleviating, suppressing, or curing a clinical skin disorder or condition.

[0082] As used herein, the term “clinical skin condition” means a clinical dermatological disorder, condition, or defect that typically manifests as damaged skin and often presents with an undesirable, unsightly skin appearance. Such skin conditions are often associated with inflammation, such as those caused by bacterial, fungal, or viral infections. Clinical skin conditions that are expected to benefit from improved skin regeneration by the cell-free preparations of the present invention or pharmaceutical or cosmetic compositions containing them include, but are not limited to, acne, actinic keratosis, atopic dermatitis, venous stasis dermatitis, eczema, basal cell carcinoma, contact dermatitis, keloids, lichen planus, melanosis, vitiligo, psoriasis, rosacea, seborrheic dermatitis, acute and chronic wounds, such as diabetic wounds, burns, erythema multiforme, epidermolysis bullosa, lupus, and keratosis pilaris.

[0083] Clinical skin conditions may also present with hyperfibrosis, which is a pathological accumulation of excessive fibrous connective tissue. This is a common response to chronic injury. Without being limited to any theory, the cell-free preparations of the present invention can reverse scarring and fibrosis, at least in scars and other fibrous areas, by inducing normal cellular function, for example, through the activation of fibroblasts.

[0084] The ability of the cell-free preparations of the present invention to reverse scarring and fibrosis is not limited to the skin as the area of ​​application. Therefore, it is assumed that the preparations are also suitable for treating other fibrous conditions other than fibrous skin conditions and hypertrophic scars, such as chronic and acute wounds, burns, and other skin injuries, keloids, and keratosis pilaris. Non-limiting examples of such other fibrous conditions include Peyronie's disease, urethral stricture, and abdominal adhesions. Accordingly, the use of the preparations for treating fibrous conditions, including not only fibrous skin conditions but also other fibrous conditions, as well as methods of such treatment in subjects requiring it, including administering the preparations in accordance with what is described elsewhere in this specification, are also provided herein.

[0085] As used herein, the term "therapeutably effective dose" means the amount that at least improves the adverse effects of a skin condition.

[0086] Those skilled in the art will readily understand that cosmetic and therapeutic treatments often overlap. Furthermore, some skin conditions can be considered both clinical dermatological conditions or defects and aesthetic skin conditions and defects. Both types of skin conditions and defects, regardless of how they are classified, benefit from treatment with the preparations of the present invention.

[0087] In some embodiments of the therapeutic and / or cosmetic uses and methods described above, the preparation or composition containing it is applied topically to skin to be treated, such as damaged skin. In some embodiments, the method may include the steps of i) preparing damaged skin by a treatment including, for example, microneedling, exfoliation, or laser resurfacing, and ii) applying the preparation or composition to the thus prepared damaged skin. In some other embodiments, the method may include the step of applying the preparation or composition to pre-damaged skin, i.e., pre-prepared skin, for example, by any of the treatments described above, and so the damaging treatment does not form part of the therapeutic and / or cosmetic treatment in some embodiments of the present invention.

[0088] As described above, when the preparations of the present invention are applied to skin areas associated with acne or other scarring that have been damaged, preferably by, for example, laser resurfacing, or preferably by any intradermal needling technique known to those skilled in the art, the healing process is accelerated, the scars fade, and a healthy skin appearance is observed. In fact, Figure 1 shows a faster healing process and improved scar reversal in response to the administration of the preparations of the present invention to a laser-resurfaced facial area associated with acne scarring, while Example 7 shows the activation of acne scar repair in response to the administration of the preparations of the present invention by intradermal injection. Furthermore, scar fading and improved skin appearance are shown in Figure 2. In conclusion, these results indicate that the preparations of the present invention have the potential to promote skin regeneration, particularly in scarred skin. Further evidence regarding the potential of the preparations to reverse scarring and dermatofibrosis is provided in Examples 8, 9, and 10. These examples demonstrate that the preparation can activate fibroblasts, induce mesenchymal stem cells, and activate keratinocytes, respectively.

[0089] Furthermore, when the preparations of the present invention are preferably applied to skin areas damaged, for example, by microneedling, a reduction in wrinkles, pores, UV spots, brown spots, and redness is observed. These results indicate that the preparations of the present invention have the potential to promote skin regeneration and / or skin rejuvenation.

[0090] In some further embodiments of the therapeutic and / or cosmetic uses and methods described above, the preparation or composition containing it is applied intradermally to skin being treated, such as acne scars, aging skin, or photodamaged skin, for example, as mesotherapy. In such embodiments, however, improvements in skin quality, health, and appearance are observed, judging by a reduction in wrinkles, pores, UV spots, brown spots, and redness. These results also indicate that the preparation of the present invention has the potential to promote skin regeneration and / or skin rejuvenation.

[0091] In one embodiment, the present invention provides the use of cell-free preparations disclosed herein for producing cosmetic compositions for non-therapeutic treatment of skin, including damaged or undamaged skin, including aged and scarred skin, and for producing pharmaceutical compositions for therapeutic treatment of clinical conditions or defects, such as clinical skin conditions.

[0092] In a further embodiment, the present invention provides the use of the cell-free preparations, cosmetic preparations, or pharmaceutical preparations disclosed herein to promote one or more of the following effects: activation of keratinocytes, activation of fibroblasts, induction of mesenchymal stem cells, and induction of endogenous production of collagen I, collagen III, collagen IV, collagen VIII, elastin, EGF, and / or MMP-1. Also provided are corresponding methods for promoting the effects, including administration of the cell-free preparations, cosmetic compositions, or pharmaceutical compositions to an area of ​​the human body where promotion of one or more of the aforementioned effects is desired. [Examples]

[0093] [Example 1] Preparation and characterization of cell-free subcutaneous tissue preparations Human aspirated adipose tissue samples were obtained from three healthy volunteers undergoing standard water-assisted liposuction, with their signed informed consent.

[0094] The aspirated adipose tissue was processed by first subjecting it to gentle shaking without rinsing, and then separating the shaken adipose tissue into an upper lipid fraction, an intermediate adipose tissue fraction containing adipocytes and other cells, and a lower aqueous fraction containing the liposuction solution used. While the intermediate fraction was removed, the lipid fraction and aqueous fraction were collected and combined, and then sterile filtered using a 0.22 μm filter. The resulting cell-free preparation was stored in aliquots at -20°C.

[0095] The three independent patches obtained in this manner from three different donors were subjected to non-targeted metabolomics analysis, a comprehensive analysis of all measurable analytes in the sample containing unknown chemicals, using a UPLC-MS platform.

[0096] Sample preparation was carried out as follows: Three batches of samples were thawed on ice, and then 100 μL of each sample was transferred to a new tube, to which 300 μL of methanol was added. Next, the samples were vortexed for 30 seconds, sonicated for 30 minutes at 4°C, and kept at -20°C for 1 hour. Then, the samples were vortexed for 30 seconds and kept at -20°C for 0.5 hours. Thereafter, the samples were centrifuged at 12000 rpm for 15 minutes at 4°C. Finally, 200 μL of the supernatant and 5 μL of DL-o-chlorophenylalanine (0.5 mg / ml) were transferred to vials for LC-MS analysis.

[0097] LC-MS analysis was performed as follows. Separation was performed using Waters Acquity UPLC in combination with Q Exactive MS (Thermo), and screening was performed using ESI-MS. The LC system consisted of ACQUITY UPLC HSS T3 (100 × 2.1 mm × 1.8 μm) and Acquity UPLC. The mobile phase consisted of solvent A (0.05% aqueous formic acid) and solvent B (acetonitrile), with gradient elution (0-1.0 min, 5% B; 1.0-12.5 min, 5%-95% B; 12.5-13.5 min, 95% B; 13.5-13.6 min, 95%-5% B; 13.6-16.0 min, 5% B). The mobile phase flow rate was 0.3 mL / min. The column temperature was maintained at 40°C, and the sample control temperature was set to 4°C.

[0098] The parameters for mass spectrometry in ESI+ and ESI- modes were as follows: ESI+: Heater temperature 300°C; Sheath gas flow rate 45 arb; Aux gas flow rate 15 arb; Sweep gas flow rate 1 arb; Spray potential 3.0 kV; Capillary temperature 350°C; S-lens RF level 30%. ESI-: Heater temperature 300°C, sheath gas flow rate 45 arb; Aux gas flow rate 15 arb; sweep gas flow rate 1 arb; spray potential 3.2 kV; capillary temperature 350°C; S-lens RF level 60%.

[0099] Based on the analysis, the sample contained over 1004 proteins, over 6000 related peptides, and over 500 metabolites. Surprisingly, neither VEGF, IGF-1, nor FGF-2 were among the identified proteins or peptides.

[0100] Instead, the samples contained various lipids, related proteins, and proteins associated with lipid metabolism; various basement membrane components, as well as proteins and peptides associated with the dermal-epidermal junction or proteins and peptides affecting the dermis and epidermis; essential and non-essential amino acids, antioxidants, vitamins, and a greater number of glycoproteins and proteoglycans, as well as factors associated with cell proliferation. Some of the identified compounds are considered endogenous, and some are considered exogenous. However, all components originate from donor tissue. It should also be noted that some components are intracellular, and some are extracellular.

[0101] Some of the identified molecules are listed in Table 1 below.

[0102] [Table 1-1] [Table 1-2] [Table 1-3]

[0103] [Example 2] Safety testing The purpose of the safety tests was to evaluate the tolerance of the same-derived aspirated adipose tissue preparation of the present invention and to identify possible medium-term adverse events (AEs) associated with its use.

[0104] To ensure uniformity of the preparations from a safety standpoint, the three independent batches disclosed in Example 1 were used in this study.

[0105] This study was conducted as a randomized, double-blind, placebo-controlled trial at a private sector hospital in Helsinki, Finland, with signed informed consent forms obtained from all participants.

[0106] To achieve this objective, a total of 38 volunteers (74% female) were recruited. Participants represented all five skin types according to the Fitzpatrick classification, with classifications 2 and 3 emphasized. 34% of the participants had some underlying medical condition, such as hypertension, migraine, or asthma; the rest were considered perfectly healthy. No participants had underlying medical conditions that could affect the interpretation of the results. Participants with a prior diagnosis of an allergic reaction leading to anaphylaxis, and those with acute cancer, were excluded from the study. Furthermore, to ensure the reliability of the results, participants were not permitted to use any medications to treat allergies or tolerances during the study.

[0107] Subcutaneous injection was chosen as the route of administration because this route is generally associated with a higher risk of adverse events (AEs) compared to topical administration. High doses (3 to 10 times the amount normally intended for use) of the test preparation were injected into the subcutaneous tissue of each subject's arm. A corresponding amount of ringer's acetate solution, acting as a placebo, was injected into the opposite arm. Ringer's acetate is not associated with AEs, is generally considered safe, and is routinely used for intravenous administration.

[0108] Study participants were monitored for three months using electronic diaries, telephone calls by trial nurses, and visits to physicians. Follow-up was initially daily, and then monthly. The study was conducted in collaboration with a clinical trial unit at a private sector hospital and monitored by an independent contract research organization (CRO).

[0109] Both allergic reactions and irritation symptoms were extensively investigated in this study. All adverse reactions were reported, evaluated, and analyzed by physicians. Participants' body temperature was measured daily for one week, and no participants reported fever associated with the administration of the test preparation. No infections at the injection site were observed during the study. Immediately after injection, the test preparation was found to cause slightly more redness at the injection site than placebo.

[0110] Based on some previous studies, the test preparation was known to cause transient vasoconstriction, i.e., pale skin, at the injection site due to vasoconstriction. This effect is related to the preparation's mechanism of action and is not considered an adverse event (AE), nor does it predict subsequent AEs. Vasoconstriction events were also reported during safety studies.

[0111] Swelling and subcutaneous bleeding were observed more frequently with placebo injections than with test injections, indicating that these reactions were related to the route of administration (injection) and fluid accumulation in the tissues. Reported acute adverse events are summarized in Table 2 below.

[0112] [Table 2]

[0113] During the 3-month follow-up period, no surprising or serious adverse events (AEs) were observed, and no abnormalities were observed at the physician's evaluation on day 90. The product was found to cause no mid-term AEs at all, particularly when evaluated for hyperpigmentation, induration, necrosis, granuloma, or other abnormalities. No significant differences were found among the three batches used in terms of AEs, confirming that the preparations of the present invention have a uniform safety profile with no batch-to-batch variability.

[0114] [Example 3] Improved overall skin healing and regeneration after laser treatment The cell-free aspirated adipose tissue-derived preparation disclosed in Example 1 was tested for its ability to improve overall skin healing and enhance scar appearance after laser resurfacing using eight volunteers (aged 22–72 years) treated in essentially the same manner as the initial subjects.

[0115] Figure 1 shows photographs of the right and left lateral profiles of a 22-year-old male whose temples and cheeks were treated with a fractional CO2 laser to reduce the appearance of acne scars. Both sides of the face were treated with the same settings. After treatment, the left side of the face was locally treated with the cell-free aspirated adipose tissue preparation of the present invention (0.5 ml) (right side photograph), while the right side of the face was treated with sterile water (left side photograph). The photographs illustrate the healing and re-epithelialization process two days after treatment. Improved recovery and faster healing were observed in the areas treated with the preparation of the present invention.

[0116] Figure 2 shows photographs of a leg that underwent four surgeries. Scars from three years prior were treated with a fractional CO2 laser using the same settings for the entire scar. The lower half of the scar was treated twice with local administration (0.5 ml) of the cell-free aspirated adipose tissue preparation of the present invention; the first administration was immediately after the laser treatment, and the second administration was performed 6 hours later. At 6 hours, vasoconstriction was observed in the scar area treated with the preparation of the present invention. Two days after laser resurfacing, the scar area treated with the preparation of the present invention showed less redness and roughness compared to the untreated area. Twenty-eight days after laser resurfacing, the scar area treated with the preparation of the present invention was much lighter in color, softer, and closer to skin level compared to the untreated scar area.

[0117] The other six subjects also reported faster recovery and less pain in the treatment area.

[0118] [Example 4] Promotion of skin regeneration in microneedling-treated skin. The cell-free aspirated adipose tissue-derived preparation disclosed in Example 1 was tested in three healthy volunteers who had undergone standard facial microneedling, a widely used cosmetic procedure that uses small sterile needles to promote collagen and elastin production in order to improve the appearance and texture of the skin, and who had given signed informed consent. The follow-up period was 4 weeks.

[0119] The first subject was a 34-year-old woman with a 13-year-old facial scar that had not shown any improvement with previous treatment using microneedling. In this study, the entire face, including the scar area, was treated with microneedling (Dermapen, 0.7mm-2mm depth), and immediately afterward, the preparation of the present invention was applied topically to the upper left side of the face (approximately 1 ml). VISIA photographs were taken before the procedure using standard angles and lighting.

[0120] At the follow-up visit four weeks after the procedure, the initial subjects reported a significant improvement in the appearance of the scar area. Before and after photographs are shown in Figure 3. At the same visit, new VISIA photographs were taken again using standard angles and lighting and compared with the previous photographs. According to the VISIA analysis, the patients showed up to 31% improvement in skin texture, pores, and redness.

[0121] Two other subjects (aged 59 and 72) were treated in essentially the same manner as the first subject. In VISIA analysis, the subjects showed up to 25% improvement in texture, pores, and redness.

[0122] All participants reported excellent results in subjective evaluations.

[0123] [Example 5] Mesotherapy treatment for aging skin Cell-free aspirated adipose tissue preparations, prepared as described in Example 1, were tested for their potential for skin regeneration and / or rejuvenation in three healthy volunteers who had given signed informed consent. The subjects had undamaged but aged skin. Each subject was treated with one of the three prepared batches.

[0124] The first subject was a 58-year-old woman treated with mesotherapy (sometimes called intradermal therapy or multi-puncture treatment). Approximately 70 papula (volume 0.04 ml / papula) were injected into the entire face. Four weeks after treatment, a significant improvement in skin texture (soft, "velvety" texture) was observed subjectively and in VISIA photographs (standard angle and light). Furthermore, the disappearance of vertical wrinkles around the mouth and eye areas was observed. According to VISIA analysis, texture and blemishes improved by up to 23%. Overall VISIA analysis showed a reduction of 2 years in skin age. Before and after photographs are shown in Figure 4.

[0125] Two other subjects (aged 67 and 70 years) were treated in essentially the same manner as the first subject. VISIA analysis showed improvements of up to 7% in texture and UV spots, and a reduction of up to 2 years in skin age.

[0126] All participants reported excellent results in subjective evaluations.

[0127] The second treatment was administered six weeks after the first. Three weeks after the second treatment, the subjects were still very satisfied and reported excellent results in their subjective assessments.

[0128] [Example 6] Intradermal injection treatment for aging skin Cell-free aspirated adipose tissue preparations, prepared as described in Example 1, were tested for their potential for skin regeneration and / or rejuvenation in 15 subjects with a median age of 53 years, using an intradermal injection procedure essentially as described in Example 5. The subjects had undamaged but aged skin. The treated areas were the face, neck, and décolleté. Subjects subjectively assessed their skin two months after the procedure. Subjects rated the sensation and appearance of their skin on a scale of 1 to 6 (1=worse than before treatment, 2=no change, 3=better than before treatment, 4=much ​​better than before treatment, 5=as expected, 6=excellent / better than expected). The results are shown in Table 3. Overall, subjects experienced very satisfactory results from the procedure, with significantly improved skin sensation and appearance compared to before treatment. In particular, patients who received décolleté treatment reported high satisfaction. When seven of the patients were re-evaluated at four months, they reported similar levels of satisfaction with skin sensation and increased satisfaction with skin appearance compared to the corresponding evaluation at two months after treatment.

[0129] [Table 3]

[0130] [Example 7] Injectable treatment for acne scars Several subjects with old acne scars were treated by injecting cell-free aspirated adipose tissue, prepared essentially as described in Example 1, into the dermis of the acne scar area to reach the full length and depth of the scarred area on the face, using techniques commonly used in acne scar injection treatments. The results were remarkably good as early as 3–7 weeks after the start of treatment. Subjects observed that the treatment with cell-free aspirated adipose tissue began to have an effect almost immediately. Dark pigmentation began to disappear, and from 3 weeks onward, the skin felt healthier, and the acne scars became less visible, shallower, and softer. At 7 weeks, the skin felt healthier and more elastic, and the scars no longer felt like hard areas on the skin. The scars became considerably softer, and stiffness with facial movement decreased. The skin surface was smoother, and the depressions and vesicles of the scars subsided.

[0131] [Example 8] Fibroblast migration and inducible ECM protein expression BJ fibroblasts (ATCC, CRL-2522) were used to test migration and modified gene expression after exposure to a cell-free aspirated adipose tissue preparation prepared essentially as described in Example 1. In both cases, BJ fibroblasts were cultured in GlutaMAX™ supplemented with MEM (Gibco, REF: 41090036) and 10% FBS (Gibco, A3160501), 1×MEM non-essential amino acid solution (Gibco, REF: 11140050), and 1% antibiotic-antifungal agent (Gibco, 15240096). For migration, 20,000 cells were seeded in each well of a 3-well cell culture insert (Ibidi, REF: 80369) to reach a confluent cell density, while qPCR was performed using cells at a density of 20,000 cells / cm³. 2Cells were seeded at a density onto 6-well Nunc® cell culture-treated multi-dishes (Thermo Scientific, REF 140685). In both experiments, cells were seeded on day -1 and a cell-free aspirated adipose tissue-derived preparation was added on day 0. In the exposure experiment, half of the total seeding volume was in complete cell culture medium. In the medium control experiment, the remaining half was in Ringer's solution, while in the substance exposure experiment, the protein concentration of the cell-free aspirated adipose tissue-derived preparation was calculated and, based on this, the cell-free aspirated adipose tissue-derived preparation was added together with Ringer's solution to a concentration of 1 mg / ml to fill the total seeding volume. For migration, two replication inserts were used for the medium control and substance exposure experiments, resulting in a total of four replications from the three-well insert, while three replications were used for qPCR.

[0132] Migration was imaged using time-laps imaging with a Leica DMi8 microscope, configured to acquire images every hour for 24 hours. The remaining areas between cell culture insert wells were measured using the Labkit plugin in ImageJ 1.54f to define cell-free regions. For clarity of results, only every 2 hours is shown in Figure 5. These results show a significant increase in fibroblast migration after exposure to the cell-free aspirated adipose tissue-derived preparation, which began at hour 4 and continued until the end of the experimental period. Increased fibroblast migration is generally known to play a role in wound healing and skin regeneration processes, and therefore these results support the ability of the cell-free aspirated adipose tissue-derived preparation to induce skin regeneration and wound healing by attracting fibroblasts to the site of tissue damage.

[0133] RNA for qPCR was purified on day 6 of exposure using the GeneJET RNA purification kit (Thermo Scientific, K0732). RNA of the same concentration was translated to cDNA from all samples using dsDNAse processing with the Maxima® H Minus cDNA synthesis master mix (Thermo Scientific, M1681). Gene expression was analyzed using TaqMan real-time PCR assays for smooth muscle actin (ACTA2), collagen 1 (COL1), collagen 3 (COL3), collagen 8 (COL8), and elastin (ELN). For the following experiments with collagen 4A (COL4A), importin 8 (IPO8) was used as the housekeeping gene in both runs, and the analysis was performed using the TaqMan® Universal PCR master mix (Applied Biosystems, 4304437). PCR was performed using the CFX96 real-time PCR system (Bio-Rad). As mentioned above, collagen and elastin are important components of the extracellular matrix (ECM) and basal layer of the dermis that support healthy skin structure, while ACTA2 is an indicator of fibroblast differentiation into myofibroblasts that produce ECM components. qPCR results (Figures 6A and 6B) show inducible expression of all measured components, supporting the ability of cell-free aspirated adipose tissue preparations to reconstruct the ECM.

[0134] [Example 9] Differentiation of human adipose-derived stem cells We modeled mesenchymal stem cells and their differentiation using primary human adipose-derived stem cells (hASCs). Differentiation was measured using the AdipoRed® (Lonza, PT-7009) triglyceride accumulation assay and qPCR gene expression analysis. Cells were cultured in DMEM / F12 supplemented with GlutaMax (Gibco, A41920-01), 10% human serum (BioSell), and 1% Antiobiotic-antimycotic (Gibco, 15240096). Both AdipoRed and qPCR measured 22,000 cells / cm³. 2For AdipoRed, cells were seeded in sets of three in a 48-well plate, and for qPCR, cells were seeded in sets of three in a 6-well plate. For qPCR, exposure was 1 mg / ml, as in Example 8, while for AdipoRed, exposure was 0.8 mg / ml and 0.4 mg / ml. Half of the total volume consisted of complete cell culture medium, which was then filled to the final volume with Ringer's solution.

[0135] AdipoRed measurements were performed according to the manufacturer's instructions. Briefly, AdipoRed was diluted to a 3% mixture using PBS (Gibco, REF). Cells were washed with RT PBS and then incubated in 3% AdipoRed for 15 minutes. Fluorescence was measured using a Tecan Spark multimode plate reader with an excitation wavelength of 485 nm and an emission wavelength of 572 nm. Cell-free aspirated adipose tissue-derived preparations induced a dose-dependent increase in triglyceride production, with both concentrations inducing a significant increase in signal (p < 0.05) (Figure 7). Triglyceride accumulation is an indicator of mesenchymal stem cell differentiation.

[0136] After 6 days of exposure to a cell-free aspirated adipose tissue preparation, RNA purification, cDNA translation, and qPCR were performed in the same manner as in Example 8. The TaqMan assays used were ACTA2 and COL1, with IPO8 used as the housekeeping gene. The results showed inductive expression of both ACTA2 and COL1 in samples exposed to the cell-free aspirated adipose tissue preparation compared to the medium control (Figure 8). These changes are indicators of hASC differentiation into specialized cell types, such as cutaneous cell types (e.g., endothelial cells and fibroblast-like cells).

[0137] The results support the potential of the cell-free preparations of the present invention for skin regeneration and anti-fibrosis.

[0138] [Example 10] Keratinocyte proliferation and migration Keratinocyte proliferation and migration were tested using pooled neonatal human epidermal keratinocytes, NHEK-neo (Lonza, 00192906). Cells were grown in KBM® Gold® basic medium (Lonza, 00192151) supplemented with KGM® Gold® SingleQuots® (Lonza, 00192152). For the proliferation assay, cells were grown at a density of 10,000 cells / cm². 2 Cells were cultured at a density on 48-Nunc®-treated multi-dishes (Thermo Scientific, 150687). For the migration assay, 40,000 cells were seeded in pairs in each well of a 3-well cell culture insert (Ibidi, REF:80369). Exposure in both the proliferation and migration assays was carried out in the same manner as in Example 8, with both exposure to the medium control and cell-free aspirated adipose tissue preparation being performed with complete cell culture medium filling half of the total exposure volume, and exposure being performed with cell-free aspirated adipose tissue preparation at a concentration of 1 mg / ml, with Ringer's solution filling up to the total volume.

[0139] Proliferation was measured on day 6 by counting nuclei stained with Hoechst 33342 (Invitrogen, H3570). Cells were washed with PBS and incubated for 10 minutes in Hoechst diluted 1:1000 in PBS. Cells were imaged using an excitation wavelength of 350 nm and an emission wavelength of 461 nm. Nuclei were calculated in ImageJ 1.54f. The results (Figure 9) show a significant increase in cell proliferation when treated with a cell-free aspirated adipose tissue-derived preparation.

[0140] On days 0, 2, 3, and 7, the progression of cell migration was imaged using a Leica DMi8 microscope. Cell-free regions of the images were analyzed using ImageJ 1.54f with the Labkit plugin. Migration was significantly increased on days 2, 3, and 7 in samples treated with cell-free aspirated adipose tissue-derived preparations (p < 0.05), and on day 7, the two cell populations from different insert sectors completely merged (Figure 10).

[0141] The cells were left on migration plates for a further 8 days, after which RNA was extracted. During cell growth, the cell culture medium and exposure were changed three times, on days 3, 7, and 10. At this point, the cell population in the medium control was still separated, while the samples treated with cell-free aspirated adipose tissue preparation were still fully combined. RNA was purified from these samples using the GeneJET RNA purification kit (Thermo Scientific, K0732). Using a method similar to that in Example 8, the RNA was treated with dsDNAse and translated to cDNA. qPCR was performed using a TaqMan assay for epidermal growth factor (EGF), matrix metalloproteinase (MMP1), and TIMP metallopeptidase inhibitor 1 (TIMP1). Matrix metalloproteinases are required for cell migration so that cells can move across the extracellular matrix, and these can be inhibited by TIMP. On the other hand, EGF induces keratinocyte proliferation and migration. qPCR analysis revealed increased gene expression of EGF and MMP1, and decreased expression of TIMP1 (Figures 11A, 11B, and 11C).

[0142] Overall, increased proliferation, migration, and gene expression of EGF and MMP1, along with decreased TIMP1 expression, indicate increased keratinocyte turnover induced by cell-free aspirated adipose tissue-derived preparations. Furthermore, EGF has various indications related to skin regeneration and the healing of skin diseases such as acne, atopic dermatitis, wounds, and scars.

[0143] [Example 11] Proteomics enrichment analysis A novel LC-MS proteomics analysis was performed using a triplicate of cell-free aspirated adipose tissue preparations derived from 10 different donors, similar to the method used in Example 1. The gene-coding proteins identified in the non-targeted proteomics analysis were further investigated by functional enrichment using the g:Profiler enrichment tool, which calculates enrichments for a gene ontology (GO) knowledge base. The enrichment analysis identified 1025 significantly enriched terms across the GO sub-ontologies: 178 molecular functions (GO:MF), 691 biological processes (GO:BP), and 156 cellular compartments (GO:CC). Subsections were performed by manually examining these terms and selecting descriptive terms by ignoring highly specific or broadly disseminated offspring and ancestral terms. This selection resulted in 25 terms, shown in Figure 12.

[0144] Overall, twelve terms were selected from GO:BP (Biological Processes). These terms include angiogenesis, tissue migration, wound healing, immune system-related processes, organization of proteolytically active cellular components, and cellular detoxification, all of which are processes relevant to tissue healing and regeneration. In GO:CC (Cellular Compartments), five terms were selected, including immunoglobulin complexes, high-density lipoprotein particles, ECM-related terms, and extracellular exosomes. These terms provide an overall overview of the constituent units in cell-free aspirated adipose tissue preparations. Finally, eight GO:MF (Molecular Function) terms were selected that highlight the antioxidant, oxidoreductase, and peptidase-modulating activity of proteins in cell-free aspirated adipose tissue preparations, as well as terms related to protein binding affinity with glycosaminoglycans, the cytoskeleton, and cell adhesion molecules.

Claims

1. A cell-free preparation derived from aspirated adipose tissue, characterized by containing at least one of the lipid fraction and aqueous fraction of aspirated adipose tissue.

2. The cell-free preparation according to claim 1, wherein the aqueous fraction comprises a used liposuction solution which may or may not contain a local anesthetic and / or adrenaline.

3. A cell-free preparation according to claim 1 or 2, comprising an aqueous fraction of aspirated adipose tissue and a lipid fraction of aspirated adipose tissue as the sole liposuction-derived material in the preparation.

4. A cell-free preparation according to claim 1 or 2, comprising, as the sole liposuction-derived material in the preparation, an aqueous fraction of aspirated adipose tissue, a lipid fraction of aspirated adipose tissue, and used liposuction solution not collected as part of aspirated adipose tissue.

5. The cell-free preparation according to any one of claims 1 to 4, wherein the preparation is filtered through a membrane filter, preferably a membrane filter having a pore size of 0.2 μm or 0.22 μm.

6. A cell-free preparation according to any one of claims 1 to 5, comprising one or more components selected from the group consisting of structural and non-structural extracellular matrix proteins, peptides, chains, subchains, and their subunits; proteins involved in the synthesis of the extracellular matrix; lipids, lipid-related proteins, proteins involved in lipid metabolism; basement membrane proteins, peptides, chains, subchains, and their subunits; specialized dermal-epidermal junction components and factors affecting the epidermis; essential and non-essential amino acids; antioxidants; vitamin derivatives and metabolites; and glycoproteins and proteoglycans.

7. i) The structural and non-structural extracellular matrix proteins, peptides, chains, subchains, and their subunits include one or more components selected from the group consisting of collagen I, III, IV, VI, XV, XVIII, fibronectin, vitronectin, elastin, hyaluronan, decorin, tenascin, laminin, lumican, and prolargin; and / or ii) The lipids, lipid-related proteins, and proteins related to lipid metabolism include one or more components selected from the group consisting of ceramides, apolipoproteins, perilipins, unsaturated and saturated fatty acids, glycerophospholipids, lysophosphatidic acid, lysophospholipids, monoglycerides, diglycerides, triglycerides, and prostaglandins; and / or iii) The basement membrane and dermal-epidermal junction proteins, peptides, chains, subchains, and their subunits include one or more components selected from the group consisting of keratin, nitrogen, versican, integrin, periplakin, and plectin; and / or iv) The vitamin derivative and metabolites include at least tretinoin, The cell-free preparation according to claim 6.

8. - A step of providing aspirated adipose tissue obtained from at least one human subject; - A step of optionally shaking the aspirated adipose tissue; - A step of removing the adipose tissue fraction of aspirated adipose tissue, including cells; - A process of filtering the remaining fraction of aspirated adipose tissue. A method for producing a cell-free preparation according to any one of claims 1 to 7, including the method described in any one of claims 1 to 7.

9. The method according to claim 8, wherein, prior to the step of removing the adipose tissue fraction, the aspirated adipose tissue is separated into an upper lipid layer, an intermediate adipose tissue layer containing adipocytes and other cells of the adipose tissue, and a lower aqueous layer.

10. - A process for providing human aspirated adipose tissue; - A step of optionally shaking human aspirated adipose tissue; - A step of discarding the adipose tissue fraction containing cells, while collecting at least one of the lipid fraction and the aqueous fraction; - The process of filtering the collected fractions A method for producing a cell-free preparation according to any one of claims 1 to 7, including the above.

11. The method according to claim 10, wherein the aspirated adipose tissue is allowed to settle before the step of collecting at least one of the lipid fraction and the aqueous fraction, so that the aspirated adipose tissue separates into an upper lipid layer, an intermediate adipose tissue layer containing adipocytes and other cells of the adipose tissue, and a lower aqueous layer.

12. The method according to any one of claims 9 to 11, wherein the step of rinsing the aspirated adipose tissue or any fraction thereof is not included.

13. The method according to any one of claims 9 to 12, wherein filtration is carried out by using a membrane filter having a pore size of 0.2 μm or 0.22 μm.

14. A cosmetic or pharmaceutical composition comprising a cell-free preparation according to any one of claims 1 to 7 and a physiologically acceptable carrier, adjuvant, and / or excipient.

15. The cosmetic or pharmaceutical composition according to claim 14, which is in the form of an ointment, lotion, cream, gel, hydrogel, oil-in-water emulsion, water-in-oil emulsion, droplets, spray, liquid, solution, powder, sustained-release or sustained-release formulation, face mask, skin patch, mousse, or foam.

16. The cosmetic composition or pharmaceutical composition according to claim 14 or 15, for topical administration or administration subcutaneously, intradermally, or into subcutaneous tissue.

17. Use of a cell-free preparation according to any one of claims 1 to 7 or a cosmetic composition according to any one of claims 14 to 16 for cosmetic treatment of the skin.

18. A non-therapeutic method for cosmetically treating skin, comprising the step of applying to the skin a cell-free preparation according to any one of claims 1 to 7 or a cosmetic composition according to any one of claims 14 to 16.

19. The use according to claim 17 or the method according to claim 18, wherein the skin is damaged skin, preferably prepared by microneedling, exfoliation, and / or laser resurfacing.

20. The use according to claim 17 or the method according to claim 18, wherein the skin is aged skin or scarred skin.

21. A cell-free preparation according to any one of claims 1 to 7 or a pharmaceutical composition according to any one of claims 14 to 16, for use in treating clinical skin conditions.

22. A cell-free preparation or pharmaceutical composition for use according to claim 21, wherein the clinical skin condition is acne, actinic keratosis, atopic dermatitis, venous stasis dermatitis, eczema, basal cell carcinoma, contact dermatitis, keloid, lichen planus, melanosis, vitiligo, psoriasis, rosacea, seborrheic dermatitis, chronic wounds, such as diabetic wounds, burns, erythema multiforme, epidermolysis bullosa, lupus, or keratosis pilaris.

23. A cell-free preparation according to any one of claims 1 to 7 or a pharmaceutical composition according to any one of claims 14 to 16, for use in treating conditions associated with fibrosis.

24. A non-therapeutic method for promoting one or more of the following effects, namely, activation of keratinocytes, activation of fibroblasts, induction of mesenchymal stem cells, and induction of endogenous production of collagen I, collagen III, collagen IV, collagen VIII, elastin, EGF, and / or MMP-1, The non-therapeutic method comprising administering a cell-free preparation according to any one of claims 1 to 7 or a cosmetic composition according to any one of claims 14 to 16 to a region of the human body where one or more of the effects described above are to be promoted.

25. Use of the cell-free preparation according to any one of claims 1 to 7 for manufacturing cosmetic compositions for non-therapeutic treatment of the skin.

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