WO2022018897A1 - Skin protective agent - Google Patents

Abstract

A filtrate of stem cells derived from adipose tissue heals wounds and protects skin from UV ray damage by promoting the production of collagen.

Description

Skin protectant

The present invention relates to a skin protectant.

The skin is a layer that covers the surface of the body, forms a boundary between the inside and outside of the body, and acts as a barrier to the external environment. The skin consists of two main layers, the epidermis (upper layer) and the dermis (lower layer), and covers a fat layer called subcutaneous fat (subcutaneous tissue).

The epidermis forms the outermost layer of the skin, prevents the evaporation of water from the skin, and plays a role of protecting the living body from the outside world. There are many epidermal basal cells on the basement membrane of the epidermis as precursor cells of epidermal keratinocytes, and the keratinocytes control the turnover of the epidermis while repeating cell proliferation in the basement membrane that constitutes the boundary with the dermis. .. In addition, as stem cells that work in an emergency such as trauma, epidermal stem cells are present in each epidermal protrusion in contact with the basement membrane.

Fibroblasts are present in the dermis and control the metabolism of extracellular matrix. Fibroblasts present in the dermis produce fibrous and hydrophilic proteins such as collagen, which can maintain the condition of the skin by imparting elasticity and strength to the skin. In addition, dermal stem cells are present near hair follicles and blood vessels.

If the skin covering the surface of the body is injured or the tissue of subcutaneous fat is lost due to injury or surgery, it is necessary to reconstruct it. In addition, the turnover of cells constituting the epidermis is delayed due to the irradiation of ultraviolet rays, the influence of dry outside air, and the deterioration of skin function due to aging, and as a result, the metabolism of the skin and the barrier function of the skin are reduced. Symptoms such as wrinkles and rough skin appear.

Wound healing agents that use regenerated cells obtained from skin reconstruction and various tissues are used to treat wounds with defective skin, and collagen production-promoting cosmetics are used to prevent wrinkles and rough skin. However, it has been studied conventionally.

Examples of therapies for wound healing include surgery including tissue removal / transplantation (Non-Patent Document 1), antibiotic therapy, physical therapy, and administration of growth factors. In recent years, regenerative medicine that restores function using stem cells for tissue and organ defects and dysfunction has attracted attention, and the ability of stem cells to regenerate themselves and differentiate into special mature cells is aligned with clinical goals. Cell therapy that is controlled in this way is being carried out.

The invention described in Patent Document 1 discloses a therapeutic composition for promoting tissue regeneration in a mammal, which comprises isolated adipose tissue-derived stem cells. When adipose tissue-derived stem cells are injected intradermally in close proximity to the treatment site, the adipose tissue-derived stem cells increase cell migration to the angiogenesis and wound site, reduce the amount of scarring, and increase tissue regeneration. This shortens the healing time of the wound and reduces the possibility of infection.

Further, the invention described in Patent Document 2 discloses a compound that inhibits γ-glutamyl transpeptidase (GGT) as a protein production promoter.

Special Table No. 2008-526762 Japanese Unexamined Patent Publication No. 2012-622666

However, the disclosures described in Patent Document 1 and Non-Patent Document 1 use adipose tissue-derived stem cells themselves for treatment for tissue regeneration, and regenerative medicine using stem cells includes up to the number of target cells required for treatment. In addition to the need to increase the number of stem cells that differentiate into, there was a risk of tumorigenesis by undifferentiated cells. In addition, it took several days to prepare the stem cells for transplantation, and it was necessary to keep the cultured cells in a state where they could differentiate, so care was taken in handling.

Recently, regenerative medicine that does not use living cells is also attracting attention. Aiming at tissue regeneration by activating or differentiating endogenous stem cells with cell proliferation and differentiation factors, the use of cytokine factors secreted from stem cells has been advocated as cell proliferation factors. However, although the regenerative effect of a cell growth factor used in regenerative medicine that does not use living cells, for example, a culture supernatant of stem cells, can be expected to be similar to that of using stem cells themselves, stem cell transplantation can be expected. It was difficult to obtain the effect exceeding the above.

Further, the disclosure described in Patent Document 2 is due to a decrease in skin function due to an external environment such as ultraviolet rays or aging by administering a compound that decomposes glutathione that reduces collagen production to fibroblasts. It suppresses the decrease in collagen production in fibroblasts, but the amount of collagen produced is limited.

Therefore, it is an object of the present invention to provide a skin protective agent that is more efficient than the conventional one, enables skin wound healing with a reduced risk of adverse events, and protects the skin from damage by ultraviolet rays and the like.

In order to solve the above problems, the present inventors focused on adult stem cells (also called tissue stem cells or somatic stem cells) and examined their effectiveness in detail. As a result, adipose-derived stem cells (ASC) , Adipose-derived regeneration cells: ADRC, Adipose-derived mesenchymal stem cells: AT-MSC, AD-MSC, etc. Filtrated Adipose Derived Regenerative Cell Lysate (also known as FADRCL) is more effective in healing skin wounds than traditional adipose tissue transplantation and concentration, and also maintains skin elasticity and makes the skin firmer. We have obtained amazing findings and results that it has the effect of protecting against the damage of ultraviolet rays.
The following inventions have been completed mainly based on the above achievements and considerations.

That is, the present invention
[1] A skin protective agent containing a filtrate of adipose tissue-derived stem cell disruption solution as an active ingredient, wherein the filtrate is one selected from the group consisting of heat shock proteins, cytokines and / or paracrine factors and cell surface proteins. A skin protectant characterized by containing the above proteins.
[2] One or more proteins selected from the group consisting of heat shock proteins, cytokines and / or paracrine factors and cell surface proteins contained in the filtrate of the adipose tissue-derived stem cell disruption solution are HSP47, HSP70, HMGB1 and Annexin A6. The skin protective agent according to [1], which comprises one or more proteins selected from the group consisting of.
[3] The skin protective agent according to [1] or [2], wherein the organism species of the adipose tissue-derived stem cells are adipose tissue.
[4] The skin according to any one of [1] to [3], wherein the skin protective agent further contains at least one of the following (a) to (c). Protective agent:
(A) Adipose tissue-derived stem cells,
(B) Crushed solution obtained by crushing adipose tissue-derived stem cells (a),
(C) A supernatant obtained by centrifuging the crushed liquid (b).
[5] A wound healing agent for promoting healing of a wound site of the skin, which comprises the skin protective agent according to any one of [1] to [4].
[6] The wound healing agent according to [5], wherein the wound is a trauma, an ischemic ulcer wound or a diabetic wound.
[7] A cosmetic containing the skin protective agent according to any one of [1] to [4].
[8] A method for producing a skin protective agent, which comprises the following steps (1) to (3):
(1) A step of crushing the frozen stem cells derived from adipose tissue by freeze-thaw treatment.
(2) The crushed solution obtained in step (1) or the supernatant obtained by centrifuging the crushed solution is filtered to obtain a filtrate, and (3) obtained in step (2). The step of formulating the filtrate.
[9] The method for producing a skin protective agent according to [8], which comprises a step of disrupting unfrozen adipose tissue-derived stem cells by ultrasonic treatment instead of the step (1).

If a skin protective agent containing the filtrate (FADRCL) obtained by filtering the disrupted solution of adipose tissue-derived stem cells (ADRC) of the present invention as an active ingredient is used, ADRC itself or the supernatant of ADRC culture (supernatant) It is also called ADRC-CM. It can promote the healing of wounds on the skin as compared with the use of "culture supernatant"), maintain the elasticity of the skin, and protect the skin from the damage of ultraviolet rays. The discovery of these new properties in FADRCL obtained by filtering the disrupted solution of ADRC, not ADRC itself, is a burden on donors and patients because the number of cells used is small. It is extremely significant because it is small, it can avoid adverse events due to cell administration, and it is not necessary to start cell culture at the timing of use. When ADRC itself is used, it takes several days (usually at least 3 days of culture) to prepare the cells, but with FADRCL, the time required for cell preparation can be shortened because it does not contain cells. , It is enough to prepare just before use. Further, by using the skin protective agent of the present invention, the amount of collagen produced by fibroblasts can be increased, and the skin can be protected from damage by ultraviolet rays and the like.

As described above, the skin protective agent of the present invention contains the filtrate (FADRCL) of the disrupted solution of adipose tissue-derived stem cells (ADRC) as an active ingredient, and is the conventional ADRC itself (that is, live cells) or the culture supernatant (ADRC). -It is completely different from the technology that uses CM).

It is a conceptual diagram of the skin tissue. It is a component diagram of a filtrate (FADRCL). It is a figure which shows the wound healing model of a mouse. It is a figure which shows the test method of the group which administered ADRC or ADRC-CM to the wound healing model of a mouse, and the control group. It is a figure which shows the comparison of the wound healing rate between the group which administered ADRC or ADRC-CM to the wound healing model of a mouse, and the control group. It is a figure which shows the comparison of the wound healing rate between the group which administered ADRC-CM or the supernatant of bone marrow-derived stem cells (BMSC-CM) to the wound healing model of a mouse, and the control group. It is a figure which shows that the crushed liquid of ADRC of a mouse or the supernatant obtained by centrifuging the crushed liquid is filtered, and the filtrate (FADRCL) is obtained. It is a figure which shows the comparison of the wound healing rate with the group which administered the filtrate (FADRCL), the group which administered ADRC, or the group which administered ADRC-CM to the wound healing model of a mouse. It is a figure which shows the collagen production effect of a filtrate (FADRCL).

Hereinafter, preferred embodiments of the present invention will be described in detail.
(Embodiment)

1. 1. Skin Protective Agent The present invention relates to a skin protectant that is more efficient than the conventional one, enables skin wound healing with a reduced risk of adverse events, and protects the skin from damage by ultraviolet rays and the like.

As shown in FIG. 1, the skin consists of two main layers, the stratum corneum and the dermis. It is composed of the "stratum spinosum" and the "basal layer", and the keratinocytes born in the basal layer move outward in sequence to become the stratum corneum, which eventually peels off. In addition, the dermis is a connective tissue having a structure in which fibroblasts, collagen, elastin and the like are spread in a complex three-dimensional manner, and brings strength, extensibility and elasticity to the skin. In addition, dermal stem cells are present near hair follicles and blood vessels.

As used herein, the term "skin protectant" is an agent for promoting the regeneration of skin tissue, and includes a wound healing agent that promotes healing of a wound site of the skin or a cosmetic that protects the skin from damage caused by ultraviolet rays.

2. 2. Skin protectants filtrate invention adipose tissue-derived stem cells (ADRC) lysate filtrate (FADRCL) as an active ingredient. The term "filter" as used herein refers to a crushed solution obtained by disrupting adipose tissue-derived stem cells, or a supernatant obtained by centrifuging the crushed solution to remove unnecessary substances and filtering the supernatant. The obtained filtrate. Removal of unwanted material does not have to be complete removal. An example of the conditions for centrifugation is 200 to 300 × g for 5 to 10 minutes.

As used herein, "included in the active ingredient" means containing an effective amount of adipose tissue-derived stem cell filtrate for treatment.

The components contained in the filtrate exert a peculiar action and effect, that is, the effect of promoting wound healing of the skin or maintaining the elasticity of the skin and protecting the skin from the damage of ultraviolet rays. This is because heat shock proteins, cytokines and / or paracrine factors (hereinafter also referred to as "paracline factors") and cell surface proteins present in the filtrate act on keratinocytes, fibroblasts, vascular endothelial cells and the like. It is thought that this is to promote skin regeneration.

For disruption of adipose tissue-derived stem cells, adipose tissue-derived stem cells (ADRC) are used, for example, in a cell suspension having a concentration range of ^{1 × 10 4} cells / ml to 1 × 10 ^{7 cells / ml.} In order to obtain a crushed solution of ADRC, ADRC may be subjected to a crushing treatment, for example, a freeze-thaw treatment or an ultrasonic treatment. The cells may be crushed by physical treatment with a French press or a homogenizer, or by non-physical treatment.

As used herein, "freezing" means placing stem cells derived from adipose tissue in an appropriate container (for example, a tube suitable for cryopreservation, a centrifuge tube, a bag, etc.) and freezing the cells. The temperature can be exemplified from -20 ° C to -196 ° C.

In the freeze-thaw treatment, the ice crystals formed by the expansion of the cells during the freezing process destroy the cells and are thawed at the time of thawing. 5 times) May be repeated. The freezing conditions in the freeze-thaw treatment are not particularly limited, but for example, freezing may be performed at -20 ° C to -196 ° C. The melting conditions are also not particularly limited. For example, melting in a water bath (for example, 35 ° C to 40 ° C), melting at room temperature, or the like can be adopted. As the cells to be subjected to the crushing treatment, not only living cells but also dead cells and damaged cells may be used. Among the crushing treatments, the freeze-thaw treatment is particularly preferable because it is simple, can avoid contamination due to contact between the device and cells, and is hygienic.

In ultrasonic treatment, unfrozen adipose tissue-derived stem cells may be subjected to ultrasonic treatment to destroy the cells. A probe that emits ultrasonic waves is immersed in a cell suspension, and the mechanical energy from the probe generates and bursts tiny bubbles, which repeatedly give a violent impact to the cells and crush them. The preferred conditions for sonication are to repeat crushing for 10 seconds and resting for 20 seconds multiple times at an output of 200 W to 300 W.

It is preferable to filter the crushed liquid or the supernatant obtained by centrifuging the crushed liquid to remove unnecessary substances. With the right pore size filter, unwanted material can be removed and sterilized at the same time. The material and hole diameter of the filter used for the filter processing are not particularly limited. However, a protein non-adsorption filter is preferable. Cellulose acetate can be exemplified as a preferable material. A metal filter may be used. The filter hole diameter is preferably 0.1 μm to 0.45 μm. It is more preferably 0.15 μm to 0.3 μm. When sterilization filtration is also performed at the same time, a pore size of 0.2 μm is preferable.

As shown in FIG. 2, the filtrate obtained by crushing ADRC contains a large amount of one or more proteins selected from the group consisting of heat shock proteins, paracrine factors, etc. and cell surface proteins in a large amount as compared with the culture supernatant. included. The filtrate containing these proteins promotes collagen production (Fig. 9), promotes skin wound healing, and exerts an effect of preventing deterioration of skin function due to skin damage from the external environment such as ultraviolet rays.

The one or more proteins contained in the filtrate include one or more proteins selected from the group consisting of HSP70, HSP47, HMGB1 and Annexin A6 (Fig. 2). Since these proteins are not cytokines secreted from cells but proteins existing in cells, they are efficiently extracted into a filtrate by disrupting ADRC and filtering.

"Heat shock protein" (Heat Shock Protein, HSP) is a group of proteins whose expression increases and protects cells when they are exposed to stress such as heat. It also manifests in other stresses such as exposure, UV radiation, and wound healing. HSP is named for each molecule according to its molecular weight. For example, HSP47 and 70 are proteins with molecular weights of 47 and 70 kDa, respectively.

"HSP47" plays a role in helping collagen fibers produced by skin fibroblasts to form triple helices and become mature collagen (Kazuhiro Nagata, "Collagen-specific molecular chaperone HSP47 and treatment for fibrotic diseases". Strategy ”, Journal of Japanese Pharmacology (FoliαPhαrmαcol. Jpn.) 121, 4-14 (2003)). Therefore, it is considered that if HSP47 is expressed in skin fibroblasts, the production amount of mature collagen is enhanced.

"HSP70" is known as a substance that resists UV damage that causes cell death.

"HMGB1" (high mobility group box-1 protein) has the function of strengthening innate immunity that promotes gene expression and gene repair, and also induces migration and proliferation of progenitor cells and stem cells to promote the repair reaction. Known as a substance to possess.

It is considered that the excellent collagen-producing effect of the filtrate is induced by the action of the heat shock protein, paracrine factor, cell surface protein, etc. contained in the filtrate (Fig. 9).

"Annexin A6" is a protein involved in signal transduction. Deficiency of AnnexinA6 increases sensitivity to mechanical stimuli, and conversely, overexpression of AnnexinA6 suppresses fast-adapting currents in sensory neurons. From this, Annexin A6 is considered to suppress pain. The pain-suppressing effect of Annexin A6 contributes to the improvement of the patient's QOL.

3. 3. Another Composition of the Skin Protective Agent In another aspect of the present invention, the filtrate contains at least one selected from the group consisting of the following (a) to (c) to constitute the skin protective agent of the present invention. You may.
(a) Adipose tissue-derived stem cells (b) Crushed solution obtained by crushing adipose tissue-derived stem cells (c) Supernatant obtained by centrifuging the crushed solution of (b)

The above (a) to (c) may be contained in the skin protective agent after isolating each of the above (a) to (c) and then mixed with the filtrate of the present invention, or administered at the same time as the filtrate of the present invention. You may.

The origin of ADRC used in the skin protective agent of the present invention, that is, the species is not limited, but considering the problem of immune rejection and the like, it is preferable to use human cells.

The skin protective agent of the present invention contains other components that are pharmaceutically acceptable, such as protective agents such as dimethylsulfoxide (DMSO) and serum albumin, antibiotics, vitamins, carriers, excipients, disintegrants, and buffers. Agents, emulsifiers, suspensions, soothing agents, stabilizers, preservatives, preservatives, saline and the like may be contained.

4. Method for Producing Skin Protective Agent The skin protective agent of the present invention can be manufactured by the following steps (1) to (3).
(1) Steps to disrupt frozen adipose tissue-derived stem cells (ADRC),
(2) The crushed solution obtained in step (1) or the supernatant obtained by centrifuging the crushed solution is filtered to obtain a filtrate (FADRCL), and (3) in step (2). Step to formulate the obtained filtrate (FADRCL)

Instead of the step (1), unfrozen adipose tissue-derived stem cells may be disrupted by sonication.
The conditions of the freeze-thaw treatment can be used in the steps (1) to (3).

The adipose tissue-derived stem cells used in step (1) may be prepared according to a conventional method. Adipose tissue-derived stem cells are widely used for various purposes, and can be easily prepared by those skilled in the art with reference to literature and books. You may use cells sold from a public cell bank, commercially available cells, or the like. Hereinafter, a method for preparing adipose tissue-derived stem cells (one example) will be described as an example of the method for preparing cells.

<Preparation method for adipose tissue-derived stem cells (ADRC)>
In the present invention, "adipose tissue-derived stem cell (ADRC)" refers to somatic stem cells contained in adipose tissue, and as long as pluripotency is maintained, the somatic stem cells are cultured (passage culture). The cells obtained by (including) also fall under the category of "adipose tissue-derived stem cells (ADRC)". Normally, ADRC is prepared in an "isolated state" as cells constituting a cell population (including cells derived from adipose tissue and other than ADRC) using adipose tissue isolated from a living body as a starting material. The "isolated state" here means a state taken out from its original environment (that is, a state that constitutes a part of a living body), that is, a state different from the original existence state by human operation. Means that you are.

ADRC is prepared through steps such as separation, washing, concentration, and culture of stem cells from the adipose substrate. The method for preparing ADRC is not particularly limited. For example, a known method (Fraser JK et al. (2006), Fat tissue: an under applied source of stem cells for biotechnology. Trends in Biotechnology; Apr; 24 (4): 150-4. Epub 2006 Feb 20. Review .; Zuk PA et al. (2002), Human adipose tissue is a source of multipotent stem cells. Molecular Biology of the Cell; Dec; 13 (12): 4279-95 .; Zuk PA et al. (2001), Multilineage cells from ADRC can be prepared according to human adipose tissue: implications for cell-based therapies. Tissue Engineering; Apr; 7 (2): 211-28. Etc.). In addition, a device for preparing ADRC from adipose tissue (for example, a Celution (registered trademark) device (Cytoli Therapeutics, USA, San Diego)) is commercially available, and the device is used to prepare ADRC. May be. Using this device, cell populations containing ADRC can be separated from adipose tissue (K. Lin. Et al. Cytotherapy (2008) Vol. 10, No. 4, 417-426). Hereinafter, a specific example of the method for preparing ADRC will be shown.

(1) Preparation of cell population from adipose tissue Adipose tissue is collected from animals by means such as excision and suction. The term "animal" as used herein includes humans and non-human mammals (including pet animals, livestock, laboratory animals; specifically, for example, monkeys, pigs, cows, horses, goats, sheep, dogs, cats, mice, etc. Rats, mammals, hamsters, etc.) are included. In order to avoid the problem of immune rejection, it is preferable to collect adipose tissue (self-adipose tissue) from the patient (recipient) to whom the skin protective agent is applied. However, it does not prevent the use of adipose tissue (allogeneic) of animals of the same species or adipose tissue of different animals.

Examples of adipose tissue include subcutaneous fat, visceral fat, intramuscular fat, and intermuscular fat. Of these, subcutaneous fat can be collected very easily under local anesthesia, so that the burden on the donor during collection is small and it can be said to be a preferable cell source. Normally, one type of adipose tissue is used, but it is also possible to use two or more types of adipose tissue in combination. In addition, adipose tissue collected in a plurality of times (not necessarily the same type of adipose tissue) may be mixed and used for subsequent operations. The amount of adipose tissue collected can be determined in consideration of the type of donor, the type of tissue, or the amount of ADRC required, and is, for example, about 0.5 to 500 g. However, considering the burden on the donor, it is preferable that the amount collected at one time is about 10 to 20 g or less. The collected adipose tissue is subjected to the following enzymatic treatment after undergoing removal and fragmentation of blood components adhering to the collected adipose tissue as necessary. The blood component can be removed by washing the adipose tissue in an appropriate buffer solution or culture solution.

Enzyme treatment is performed by digesting adipose tissue with enzymes such as collagenase, trypsin, and dispase. Such enzyme treatment may be carried out by a method and conditions known to those skilled in the art (for example, R.I. Freshney, Culture of Animal Cells: A Manual of Basic Technique, 4th Edition, A John Wiley & Sones Inc., See Publication). The cell population obtained by the above enzymatic treatment includes pluripotent stem cells, endothelial cells, stromal cells, blood cell lineage cells, and / or progenitor cells thereof. The types and proportions of cells that make up the cell population depend on the origin and type of adipose tissue used.

(2) Acquisition of sedimented cell population (SVF fraction: stromal vascular fractions) The cell population is subsequently subjected to centrifugation. The sediment from centrifugation is collected as a sedimented cell population (also referred to as "SVF fraction" in the present specification). The conditions for centrifugation vary depending on the type and amount of cells, but are, for example, 1 to 10 minutes and 800 to 1500 rpm. Prior to the centrifugation, it is preferable that the cell population after the enzyme treatment is subjected to filtration or the like to remove the enzyme-undigested tissue or the like contained therein.

The "SVF fraction" obtained here includes ADRC. The type and ratio of cells constituting the SVF fraction depend on the origin and type of adipose tissue used, the conditions of enzyme treatment, and the like. In addition, the pamphlet of International Publication No. 2006/006692A1 shows the characteristics of the SVF fraction.

(3) Selective culture of adhesive cells (ADRC) and cell recovery The SVF fraction contains ADRC and other cell components (endothelial cells, stromal cells, blood cell lineage cells, precursor cells thereof, etc.). .. Therefore, in one aspect of the present invention, the following selective culture is performed to remove unnecessary cellular components from the SVF fraction. Then, the cells obtained as a result are used as ADRC in the present invention.

First, the SVF fraction is suspended in an appropriate medium, then sown in a culture dish and cultured overnight. Floating cells (non-adhesive cells) are removed by media exchange. After that, the culture is continued while appropriately exchanging the medium (for example, once every 2 to 4 days). Perform subculture if necessary. The number of passages is not particularly limited, but it is not preferable to repeat the passages excessively from the viewpoint of maintaining pluripotency and proliferative ability (preferably limited to about 5 passages). As the culture medium, a normal animal cell culture medium can be used. For example, Dulbecco's modified Eagle's Medium (DMEM) (Nissui Pharmaceutical Co., Ltd., etc.), α-MEM (Dainippon Pharmaceutical Co., Ltd., etc.), DMEM: Ham's F12 mixed medium (1: 1) (Dainippon Pharmaceutical Co., Ltd., etc.) , Ham's F12 medium (Dainippon Pharmaceutical Co., Ltd., etc.), MCDB201 medium (Functional Peptide Laboratory), etc. can be used. A medium supplemented with serum (fetal bovine serum, human serum, sheep serum, etc.) or serum substitute (Knockout serum replacement (KSR), etc.) may be used. The amount of serum or serum substitute added can be set, for example, in the range of 5% (v / v) to 30% (v / v).

Adhesive cells selectively survive and proliferate by the above operations. Subsequently, the proliferated cells are collected. The recovery operation may be performed according to a conventional method, and cells after enzymatic treatment (trypsin or dispase treatment) can be easily recovered by exfoliating them with a cell scraper or a pipette. Further, when the sheet is cultured using a commercially available temperature-sensitive culture dish or the like, it is possible to collect the cells as they are in the form of a sheet without enzyme treatment. By using the cells (ADRC) recovered in this way, a cell population containing ADRC with high purity can be prepared.

(4) Low-serum culture (selective culture in low-serum medium) and cell recovery In one aspect of the present invention, the following low-serum culture is performed in place of the above-mentioned operation (3) or after the above-mentioned (3) operation. conduct. Then, the cells obtained as a result are used as ADRC in the present invention.

In low serum culture, the SVF fraction (using the cells recovered in (3) when performing this step after (3)) is cultured under low serum conditions and the desired pluripotent stem cells (ie, ADRC). ) Is selectively propagated. Since the low serum culture method requires only a small amount of serum, when the ADRC obtained by the method of the present invention is used for therapeutic purposes, the serum of the subject (patient) itself can be used. That is, it is possible to culture using self-serum. The "low serum condition" here is a condition in which 5% or less of serum is contained in the medium. Cell culture is preferably carried out in a culture medium containing 2% (V / V) or less of serum. More preferably, cells are cultured in a culture medium containing 2% (V / V) or less serum and 1 to 100 ng / ml fibroblast growth factor-2 (bFGF).

When used for human treatment, the serum is preferably human serum, more preferably serum to be treated (that is, autologous serum).

As the medium, a normal medium for culturing animal cells can be used, provided that the amount of serum contained at the time of use is low. For example, Dulbecco's modified Eagle's Medium (DMEM) (Nissui Pharmaceutical Co., Ltd., etc.), α-MEM (Dainippon Pharmaceutical Co., Ltd., etc.), DMEM: Ham's F12 mixed medium (1: 1) (Dainippon Pharmaceutical Co., Ltd., etc.), Ham's F12 medium (Dainippon Pharmaceutical Co., Ltd., etc.), MCDB201 medium (Functional Peptide Laboratory), etc. can be used.

Pluripotent stem cells (ADRC) can be selectively proliferated by culturing by the above method. In addition, since pluripotent stem cells (ADRC) that proliferate under the above culture conditions have high proliferative activity, the number of cells required for the present invention can be easily prepared by subculture. In addition, the pamphlet of International Publication No. 2006/006692A1 shows the characteristics of cells that selectively proliferate by culturing the SVF fraction with low serum.

Subsequently, the cells selectively proliferated by the above low serum culture are collected. The collection operation may be performed in the same manner as in the case of (3) above. By using the recovered cells (ADRC), a cell population containing ADRC with high purity can be obtained.

In the above method, cells grown by culturing the SVF fraction with low serum are used, but the cell population obtained from adipose tissue is directly (via centrifugation to obtain the SVF fraction). Cells proliferated by low serum culture may be used as ADRC. That is, in one aspect of the present invention, cells proliferated when a cell population obtained from adipose tissue is cultured at low serum are used as ADRC. Further, instead of the pluripotent stem cells obtained by selective culture ((3) and (4) above), the SVF fraction (containing adipose tissue-derived stem cells) may be used as it is. The term "used as it is" here means that it is used in the present invention without undergoing selective culture.

5. Uses of Wound Treatment Agents As used herein, a "wound" is a disease, disorder, syndrome, abnormality, pathology of the skin and / or lower connective tissue, such as post-surgical skin trauma, skin contusion due to mechanical trauma. Includes all disorders characterized by erosive or burns, mucosal wounds after infection or drug treatment, diabetic wounds, skin trauma after transplant surgery and vascular regrowth after angiogenesis.

As used herein, the term "therapeutic agent" refers to a drug that has a therapeutic effect on wounds and skin dysfunction. The therapeutic effects include alleviating the symptoms (pathological conditions) or associated symptoms characteristic of physical damage to the skin such as wounds and pressure ulcers (mitigation), and preventing or delaying the worsening of the symptoms. The latter can be regarded as one of the preventive effects in terms of preventing the deterioration of skin function. Typical of the prophylactic effect is to prevent or delay the recurrence of symptoms characteristic of the target disease. In addition, as long as it shows some therapeutic effect, preventive effect, or both of them, it corresponds to a therapeutic agent for a target disease.

The wound healing process is broadly divided into three stages: inflammatory reaction stage, proliferation stage, and reconstitution stage. In this process, heat shock proteins, paracrine factors, and the like act on keratinocytes, fibroblasts, endothelial cells, and the like to promote skin regeneration. Hereinafter, it will be described in detail.

When the skin is damaged, various chemicals are released from the platelets during blood coagulation and the cell membrane of the destroyed cells during the inflammatory reaction phase, and these chemicals permeate the surrounding tissues and cause abnormalities from macrophages. A signal that it has happened is sent.

Next, in the proliferative phase, the substance released from macrophages stimulates fibroblasts to be attracted to produce collagen, which is the main material for repair. In addition, the capillaries develop supported by collagen produced from fibroblasts, and the fresh blood flowing into the capillaries supplies nutrients and oxygen to the fibroblasts, further promoting the production of collagen, which is a cycle of self-proliferation. It is composed. Thus, collagen plays an important role in the wound healing process.

By the way, regarding the effect of adipose tissue-derived stem cell (ADRC) administration on wound healing, it is said that there are two systems. One is a system in which wounds heal by differentiating ADRC itself into various cells. The other is that various paracrine factors secreted from ADRC act on various cells to promote skin regeneration, or paracrine factors and the like exert anti-inflammatory effects to promote wound healing. It is a system. Regarding the effect of paraclaline factor and the like on various cells, it has been proposed for stem cells other than adipose tissue-derived stem cells such as bone marrow-derived stem cells. However, so far, it has been fully clarified which stem cells show the most effective action, what concentration is the most effective, and what kind of combination can be used. There wasn't.

The present inventors compared adipose tissue-derived stem cells with other stem cells such as bone marrow-derived stem cells using a mouse wound healing model, and discovered the superiority of adipose tissue-derived stem cells (ADRC). Further, the filtrate of the adipose tissue-derived stem cell disruption solution (FADRCL) was compared with the culture supernatant (ADRC-CM) containing the adipose tissue-derived stem cells (ADRC) itself and the secretions of the adipose tissue-derived stem cells, and the filtrate of the present invention was compared. Discovered the superiority of (FADRCL). It will be described in detail in Examples.

<Administration method>
The skin protectant used for wound healing is usually administered to patients with wounds and pressure ulcers, but the skin protectant may be used for experimental or research purposes to confirm and verify its effect.

The skin protectant used for wound healing is preferably administered by local injection or application to the affected area. The injection or application site is typically a wound or pressure ulcer site, but may be injected or applied to the periphery thereof. In addition, it may be administered to two or more injection sites or application sites at the same time or at intervals of time.

An example of the dose (injection or application amount) of the skin protective agent is as follows. For example, ^{a skin protective agent containing a filtrate prepared from 1 × 10 5} cells / ml to 1 × 10 ⁶ cells / ml adipose tissue-derived stem cells. Examples thereof are 0.5 cc to 2.0 cc, preferably 0.8 cc to 1.5 cc. Instead of administering the entire dose at one time, the injection or application site may be staggered and administered in multiple doses.

The administration schedule may be created in consideration of the gender, age, body weight, pathological condition, etc. of the subject (patient). In addition to a single dose, multiple doses may be administered continuously or periodically. The administration interval when multiple doses are administered is not particularly limited, and 1 to 14 days can be exemplified. Further, the number of administrations is not particularly limited. The number of administrations can be exemplified once to four times a day. Examples of the administration method include a method of locally injecting or applying as an ointment to the wound site, and a method of spraying as a liquid or powder.

6. Uses of cosmetics

As shown in FIG. 1, the skin consists of an epidermis composed of epidermal keratinocytes, extracellular matrix such as collagen and elastin (extracellular matrix (hereinafter also referred to as “ECM”)), and fibers producing them. It consists of the dermal skin, which is composed of blast cells, and plays various roles such as protecting the inside of the living body and regulating body temperature. The skin is supported by ECM, and when the skin function decreases with aging and the ECM expression level decreases, it causes wrinkles and sagging. Conventionally, as a method for improving this skin symptom, a cosmetic containing collagen has been directly applied to the skin, but the effect has been transient.

Fibroblasts present in the dermis of the skin produce extracellular matrix (ECM) such as collagen and elastin. Therefore, it is important to enhance the collagen-producing ability of fibroblasts to maintain skin tension and prevent wrinkles and sagging.

As mentioned above, HSP47 (heat shock protein 47) plays a role in helping collagen fibers produced by fibroblasts to form a triple helix and become mature collagen. Therefore, if HSP47 is expressed in fibroblasts, the production of mature collagen is considered to be enhanced (Patent Document 2. Kazuhiro Nagata, "Collagen-specific molecular chaperone HSP47 and fibroblast treatment strategy", Nikkei Journal. 121.4-14 2003).

Ultraviolet rays (UV) that damage the skin are classified into UVA (320-400nm), UVB (290-320nm), and UVC (100-290nm) according to the difference in wavelength. Among them, UVB is on the upper part of the dermis. It is known to reach, be highly cytotoxic, and cause photoaging on the skin.

Sunscreen agents are generally used to suppress photoaging. Since sunscreens block UV rays that cause photoaging, they can be expected to be highly effective as a countermeasure against photoaging, but if the sunscreens run off due to sweat, etc., the skin becomes unprotected. was there.

HSP70 (heat shock protein 70) is said to have a skin protection (cell protection) effect, an inflammatory reaction suppression effect, and a DNA damage suppression effect. If HSP70 is expressed in epidermal cells, UVB-induced skin damage and cell death may be suppressed (Matsuda Minoru, "Protective effect of HSP70 against photoaging", Kumamoto University Graduate School of Pharmaceutical Education, Molecular Function Pharmacy Major: Medicinal Chemistry Course, Pharmacy and Microbiology, 2011 Doctoral Thesis, etc.).

HSP70 can be used with an activator that increases its intracellular concentration and / or activity. Examples of the activator include small molecule inducers of heat shock proteins, hydroxylamine derivatives (eg, bimochromol, arimochromol, iroxanadine and BGP-15) and the like.

<Administration method>
In addition to being applied to the skin as a skin protective agent and used, the dosage form as a cosmetic, a bathing agent, or a subcutaneous injection agent can be arbitrarily selected according to the purpose. In particular, it is preferably widely used as a cosmetic, and can be in the form of a cream, ointment, milky lotion, solution, gel, powder, granule or the like, or in the form of a pack, lotion, powder, stick or the like. As pharmaceuticals and the like, it can be widely used in the form of subcutaneous injections, ointments and the like.

Hereinafter, the use of the wound healing agent and cosmetics of the skin protective agent of the present invention will be described in more detail with reference to examples.

1. 1. Content of HSP70, HSP47, HMGB1 and Annexin A6 in the filtrate

<Test 1>
The filtrate prepared by the method described herein using normal human skin fibroblasts (NHDF; Lonza) was used at ^{a concentration of 1 × 10 6} cells / ml of adipose tissue-derived stem cells. A cellulose acetate filter having a pore size of 0.2 μm was used for the filtering treatment. In the freeze-thaw treatment, freeze-thaw was performed three times by the same method, and each component was measured each time. In order to compare with the component amount of the filtrate, the supernatant of the medium being cultured was used as the culture supernatant, and the component amount was measured. All measurement procedures were performed at room temperature.

<Quantitative HSP70>
The content in the filtrate was measured using the Human HSP70 ELISA Kit BMS2087 (invitrogen) (hereinafter referred to as “Kit” in this section).
(1) 50 μl of Sample Diluent and 50 μl of filtrate were placed in a well on a microplate in the Kit. Standard dilution was adjusted according to the instructions attached to the Kit.
(2) Covered with an adhesive film and left at room temperature for 2 hours.
(3) The liquid in the well was discarded, 400 μl of Wash Buffer was put into the well, and washed 6 times.
(4) 100 μl of Biotin-Conjugate was added, covered with an adhesive film, and left at room temperature for 1 hour.
(5) The liquid in the well was discarded, 400 μl of Wash Buffer was put into the well, and washed 6 times.
(6) 100 μl of Streptavidin-HRP was placed in a well, covered with an adhesive film, and left at room temperature for 30 minutes.
(7) The liquid in the well was discarded, 400 μl of Wash Buffer was put into the well, and washed 6 times.
(8) 100 μl of TMB Substrate Solution was placed in a well and left at room temperature for 30 minutes.
(9) 100 μl of Stop Solution was put into the well.
(10) Absorbance was measured at a wavelength of 450 nm with a microplate reader.
(11) From the measurement result of Standard, a calibration curve was drawn, and the absorbance value of the filtrate was substituted into the formula of the calibration curve to obtain the concentration.

<Quantitative determination of HSP47>
The content in the filtrate was measured using Heat Shock Protein 47 (SERPINH1) Human ELISA Kit ab108861 (hereinafter referred to as “Kit” in this section).
(1) Standard, zero control and 50 μl of filtrate were placed in a microplate in the Kit.
(2) In (1), 50 μl of Assay Buffer was added per well, and the mixture was sealed and left for 2 hours.
(3) The liquid in the well was discarded, 200 μl of 1 × Wash Buffer was put in the well, washed 5 times, and completely wiped off with an absorbent paper towel.
(4) 50 μl of HSP47-antibody conjugate was put in a well, sealed, and left at 37 ° C for 2 hours.
(5) 50 μl of 1 × SP Conjugate was placed in a well and left at room temperature for 30 minutes.
(6) Absorbance was measured at a wavelength of 450 nm with a microplate reader.
(7) From the measurement result of Standard, a calibration curve was drawn, and the absorbance value of the filtrate was substituted into the formula of the calibration curve to obtain the concentration.

<Quantitative HMGB1>
The content in the filtrate was measured using the HMGB1 ELISA Kit ARG81351 (Arigo Biolaboratories Corporation) (hereinafter referred to as "Kit" in this section).
(1) Standard, zero control and 50 μl of filtrate were placed in a microplate in the Kit.
(2) 50 μl of Assay Buffer per well was added to (1), sealed, and left at 4 ° C. overnight.
(3) The liquid in the well was discarded, and 350 μl of chilled 1 × Wash Buffer was placed in the well and washed 3 times.
(4) A 100 μl 1 × HRP-antibody conjugate was placed in a well, sealed, and left at 37 ° C for 1 hour.
(5) (3) was repeated.
(6) 100 μl of 1 × TMB substrate was placed in a well and left at room temperature for 10 minutes.
(7) 50 μl of 1 × Stop solution was put into the well.
(8) Absorbance was measured at a wavelength of 450 nm with a microplate reader.
(9) From the measurement result of Standard, a calibration curve was drawn, and the absorbance value of the filtrate was substituted into the formula of the calibration curve to obtain the concentration.

<Quantitative determination of Annexin A6>
The content in the filtrate was measured using a Human Annexin A6 (ANXA6) ELISA Kit EK12650 (Signalway Antibody) (hereinafter referred to as “Kit” in this section).
(1) Put 100 μl of Standard and the filtrate per 1 well in the microplate in the Kit.
(2) Sealed and left at 37 ° C for 2 hours.
(3) Discard the liquid in the well.
(4) 100 μl of Detection Reagent A working solution was put into a well, sealed, and left at 37 ° C. for 1 hour.
(5) The liquid in the well was discarded, 300 μl of 1 × Wash Solution was put into the well, and washed 3 times.
(6) 100 μl of Detection Reagent B working solution was put into a well, sealed, and left at 37 ° C. for 1 hour.
(7) The operation of (5) was repeated 5 times.
(8) 90 μl of Substrate Solution was placed in a well and left at 37 ° C for 15 to 25 minutes.
(9) 50 μl of Stop Solution was put into the well.
(10) Absorbance was measured at a wavelength of 450 nm with a microplate reader.
(11) From the measurement result of Standard, a calibration curve was drawn, and the absorbance value of the filtrate was substituted into the formula of the calibration curve to obtain the concentration.

<Protein quantification>
The content in the filtrate was measured using the PierceTM BCA Protein Assay Kit 23227 (ThermoFisher) (hereinafter referred to as "Kit" in this section).
(1) Add 25 μl of Standard and filtrate per well to 96-Well Plate. The filtrate used was 1 × 10 ⁶ pieces / mL concentration.
(2) 200 μl of Working Reagent (50: 1; Reagent A: B) was put in a well and shaken for 30 seconds.
(3) Covered and left at 37 ° C for 1 hour.
(4) The temperature was returned to room temperature, and the absorbance was measured at a wavelength of 595 nm with a microplate reader.
(5) From the measurement results of Standard, a calibration curve was drawn, and the absorbance value of the filtrate was substituted into the formula of the calibration curve to obtain the concentration.

The contents of the above components in the filtrate (FADRCL) are shown in Table 1. As shown in FIG. 2, when the filtrate (FADRCL) and the culture supernatant (ADRC-CM) are compared, it can be seen that the filtrate contains a large amount of heat shock protein, paracrine factor and the like. The difference is that the filtrate (FADRCL) disrupts adipose tissue-derived stem cells (ADRC) to extract components of the cell contents, whereas the culture supernatant (ADRC-CM) is from adipose tissue-derived stem cells (ADRC). It is considered that this is due to the fact that it is a secretion, and the difference in the amount of various components between the filtrate (FADRCL), which is the active ingredient of the present invention, and the culture supernatant (ADRC-CM) has been clarified. The contents of HSP70, HSP47, HMGB1 and Annexin A6 per 1 ml of filtrate or culture supernatant were divided by the amount of protein per 1 ml of filtrate or culture supernatant to enable strict comparison.

2. 2. Uses of therapeutic agents for wound sites Using a mouse wound healing model, adipose tissue-derived stem cell (ADRC) transplantation during the wound healing process and culture supernatant after culturing adipose tissue-derived stem cells (conditioned-medium (ADRC-CM)) ) Was examined for its effect and mechanism. Then, it was compared with bone marrow-derived stem cells (hereinafter also referred to as "BMSC") and evaluated for the superiority of ADRC.

(1) Preparation of non-cell preparation FADRCL ADRC was collected and cultured by a conventional method from balck6 wild type mice. After adjusting the concentration (1 × 10 ⁶ pieces / ml PBS), the cells were stored overnight at -30 ° C, and the cell fluid was thawed at room temperature. After crushing the cells in this way, the crushed solution was centrifuged (290 × g for 10 minutes), and the supernatant was collected. Next, the supernatant of the crushed solution was filtered through a cellulose acetate membrane filter (pore size 0.2 μm) to obtain a filtrate (FADRCL). Moreover, in order to compare with the component amount of the filtrate (FADRCL), the component amount of the culture supernatant (ADRC-CM) which is the supernatant of the medium was measured.

(2) Creation of a wound healing model for mice As shown in Fig. 3, after removing hair from the back of the mouse, a circular skin biopsy punch with a diameter of 8 mm and a depth corresponding to a full-thickness defect in the skin was used. One wound was created. After the wound was created, the prepared preparation was applied to the wound portion, and the wound portion and its surroundings were extensively covered with a covering film.

(3) Changes in wound healing rate over time <Test 2 Verification of ADRC effect and administration method>
A. As shown in Fig. 4, in Test 2, in order to verify the effect of ADRC on wound healing and the administration method, the ADRC group, the conditioned-medium group (ADRC-CM group), which is the culture supernatant after culturing ADRC, and A comparative study was conducted in three groups of the control group. The ADRC-CM group was added in order to examine the effects of non-cell pharmaceuticals, especially paraclinic factors, rather than cellular pharmaceuticals.

ADRC was collected from balck6 wild type mice, cultured, and administered to the target mice after matching the cell count and other conditions. The target mice used were 8-week male wild type mice, 5 in each group, for a total of 15 mice. The detailed conditions for each group are as follows: In the ADRC group, 1 × 10 ⁵ cultured ADRCs were used, dissolved in 200 μl of medium, and then the gelling agent (KOKEN collagen acidic solution I-PC 5 mg / ml pH 3.0) was used. Sterile Atelocollagen Acidic Solution) 200 μl was mixed and administered as a gel-like preparation. In the ADRC-CM group, the medium was changed 2 days before administration. At that time, the ratio was adjusted to 200 μl of medium with respect to ⁵ ADRCs 1 × 10, and the cells were cultured for 48 hours. After 48 hours, 200 μl of the gelling agent was mixed with 200 μl of DMEM and applied to mice. In the control group, only 200 μl of medium was mixed with 200 μl of gelling agent (Table 2).

The method of administration to mice was unified as a gel-like preparation. After applying the gel-like preparation with a spatula to the skin defect of the mice of each group, the wound and its surroundings were extensively covered with a covering film. Considering that the effect may not be sufficient with only one dose, frequent doses were given on Days 0, 1, 3, 5, and 7 and observed until the 14th. The wound healing process was followed and evaluated for 2 weeks.

B. Results The treatment evaluation method used macroscopic changes in wound area. After treatment, the wound was photographed regularly and the area of the wound was measured.
In FIG. 5, the area on each evaluation day is shown as a percentage with the wound area on the treatment day as a reference value, and the horizontal axis is the passage of time and the vertical axis is the wound area ratio. Compared with the control group, the wounds were significantly reduced in both the ADRC group and the ADRC-CM group. Although there was no difference when the ADRC group and ADRC-CM group were directly compared, it was suggested that the treatment with ADRC-CM had the same therapeutic effect as the treatment with ADRC. From this result, it was suggested that not only the effect of ADRC cells themselves but also the paracrine factor secreted from them may be important in wound healing.

<Test 3 Comparison of effects with bone marrow-derived stem cells (BMSC)>
Next, the therapeutic effects of ADRC and BMSC, which is a stem cell other than ADRC, were compared. In Test 2, it is suggested that various paraclinic factors secreted from ADRC may act on various cells to promote skin regeneration and promote wound healing by exerting an anti-inflammatory effect. Was done. Therefore, in Test 3, it was decided to compare the culture supernatant (conditioned medium) without using the cells themselves.

As shown in Table 3, ADRC and bone marrow-derived stem cells (BMSC) were collected, isolated, and cultured from mice in the same manner as in Test 2. BMSC was collected from the femur of a mouse and isolated. In the ADRC-CM group, the medium was changed 2 days before administration, as in Test 2. At that time, ^{the ratio of medium 200 μl to 5} ADRC 1 × 10 was adjusted, and the cells were cultured for 48 hours. After 48 hours, 200 μl of gelling agent was mixed with medium and applied to mice. In the BMSC-conditioned medium (BMSC-CM) group, the cells were cultured under the same conditions and cell number, and applied to mice as a gel-like preparation. As for the control group, only 200 μl of medium was mixed with 200 μl of the gelling agent as in Test 1 (Table 3). After that, the test was continued with the same protocol as in Test 2, and the healing process of the wound was followed and evaluated.

B. Results The treatment evaluation method used macroscopic changes in wound area. After treatment, the wound was photographed regularly and the area of the wound was measured. As in Test 2, when the wound area on the day of treatment was 100%, the wound area on each evaluation day was taken as a percentage.
As shown in FIG. 6, the ADRC-CM group showed a significant wound reduction effect as compared with the control group. The BMSC-CM group also showed some reduction in the wound area, but when comparing ADRC-CM and BMSC-CM, ADRC-CM showed a significant reduction in the wound area on Days 5, 7, and 9. rice field. Overall, there was a tendency for the wound to shrink in ADRC-CM compared to BMSC-CM. In other words, it was suggested that treatment with ADRC-CM was more effective than treatment with BMSC-CM.

From the results of Test 3, it was suggested that there was a difference in the secreted paraclinic factors, etc. between ADRC and BMSC, which caused a difference in the effect on wound healing. That is, ADRC is a cell that attaches to adipose tissue, has high proliferative capacity, and is excellent in differentiation into bone and cartilage and secretion of humoral factors, whereas BMSC is a cell that circulates in the blood. It has the property of differentiating into nerve cells, and it is thought that the two are completely different. Therefore, the present inventors decided to pay attention to the paracrine factor of ADRC and the like.

<Effect of Test 4 Adipose Tissue-Derived Stem Cell Filtration (FADRCL)>
From the results of Test 2 and Test 3, the present inventors examined a more effective treatment method using ADRC, and focused on FADRCL, which is one of the non-cell preparations.
As shown in FIG. 7, this pharmaceutical product is prepared by collecting and culturing ADRC from mice in the same manner as in Test 1 and Test 2, collecting cells, and then repeating freeze-thaw pulverization and filtration with a filter according to the above-mentioned method. To. It is considered that this filtrate contains a large amount of stem cell-derived paracrine factor and the like.

Here, the difference in treatment of the wound site using ADRC and FADRCL will be explained. Since the treatment method in which ADRC itself is administered is to heal the wound by engrafting / differentiating the ADRC cells at the wound site, it is essential that the ADRC cells are sufficiently engrafted / differentiated. When ADRC cells are sufficiently engrafted / differentiated, a synergistic effect with paracrine factors secreted from ADRC cells can be expected, but when ADRC cells cannot be sufficiently differentiated, the expected therapeutic effect can be obtained. May not be.

On the other hand, in the treatment method using ADRC-CM, since the paracrine factor secreted from ADRC is used, adverse events due to cell administration can be avoided, and the treatment using the cells themselves is possible. Compared to the method, it is possible to prepare a large amount of therapeutic agent in advance. Similar to ADRC-CM, FADRCL also uses paracrine factors secreted from ADRC cells, but FADRCL is produced by crushing ADRC, so in addition to paracrine factors, etc. , Cell internal and surface proteins are also believed to be contained, which may lead to anti-inflammatory effects and promote wound healing. As described above, FADRCL is considered to contain a large amount of paracrine factor and various proteins as compared with ADRC-CM.

Therefore, we decided to verify the effect of FADRCL on wound healing.
As shown in Table 4, ADRC was collected and cultured from mice using the same protocol as in Test 2, and a preparation to be administered to each group was prepared. In the FADRCL group, ADRC 1.0 × 10 ⁵ pieces were treated to prepare a filtrate, mixed with 200 μl of medium and 200 μl of gelling agent, and applied to mice. In the ADRC-CM group and ADRC group, the preparations were prepared under the same conditions as in Test 2 and Test 3, and the conditions for each group were unified (Table 4). According to the same protocol as in Test 2 and Test 3, each preparation was applied to mice, and the healing process of the wound was followed and evaluated.

B Results Figure 8 shows the time course of the wound healing rate. As in Test 2 and Test 3, the wound area on each evaluation day is used as a percentage when the wound area on the treatment day is 100%.
As shown in FIG. 8, no difference was observed between the ADRC group and the ADRC-CM group, but the therapeutic effect was similar. On the other hand, when the FADRCL group was compared with the ADRC group and the ADRC-CM group, no difference was observed 1 to 3 days after the treatment, but the wound site was significantly reduced after 5 days. In other words, it was found that the treatment with FADRCL was more effective than the treatment with ADRC and ADRC-CM.

(4) Summary As described above, the present inventors confirmed in Test 2 that various paraclinic factors secreted from ADRC-CM act on various cells to promote skin regeneration, and tested. In 3, we discovered the superiority of ADRC-CM by comparison with BMSC-CM among stem cells, and focused on various paraclinic factors secreted from ADRC-CM, and in Test 4, they are contained in a large amount in FADRCL. We have discovered the effects of various proteins and paracrine factors on promoting wound healing, and completed the invention of use of the wound healing agent of the present invention.

3. 3. Use as cosmetics (1) Promotion of collagen production <Test 5 Collagen production effect of FADRCL (freeze-thaw crushing)>
A 12-well plate was inoculated with ^{1 × 10 5} normal human skin fibroblasts (NHDF; Lonza) per well. Mesen PRO RS was used as the medium. 37 ° C., and cultured under CO ₂ 5% of the environment. When the cells became subconfluent, the medium was removed, the medium was changed to the following, the culture was continued, and the supernatant was collected after 24 hours.
(1) Medium (medium 500 μl) only (no filtrate)
(2) Medium (Medium500myueru) + filtrate 5% (human adipose stem cells 2.5 × 10 ⁵ cells equivalent)
(3) medium (Medium500myueru) + filtrate 10% (human adipose stem cells of 5 × 10 ⁵ cells equivalent)

The amount of collagen produced was measured using Human Collagen type I, ELISA kit. Absorbance was measured at a wavelength of 450 nm. A calibration curve was drawn from the measurement results of Standard, and the absorbance value of the filtrate was substituted into the formula of the calibration curve to calculate the amount of collagen of interest. In addition, in order to compare the amount of collagen produced with the amount of protein produced as a whole, the amount of protein produced was also calculated by the above method.

B. Results Table 5 shows the results of collagen and protein production after 24 hours. It was revealed that FADRCL has a concentration-dependent effect of inducing collagen production in skin fibroblasts (Fig. 9).

(2) Content of heat shock protein The contents of HSP47 and HSP70 in the filtrate are as shown in Table 1 and FIG. It was found that the filtrate (FADRCL) obtained by freeze-thawing and crushing ADRC and filtering it contained a large amount of HSP47 as compared with ADRC-CM (Table 1, FIG. 2). This suggests that HSP47 plays a role in helping collagen fibers produced by fibroblasts to become mature collagen, and thus contributes significantly to the induction of collagen production together with paraclinic factors and the like. Collagen.

HSP70 is said to have a skin protection (cell protection) effect, an inflammatory reaction suppression effect, and a DNA damage suppression effect. As shown in Fig. 2, the content of HSP70 in the filtrate is particularly high, and UVB-dependent skin damage and cell death may be suppressed by application to the epidermis (Matsuda Minoru, "Protection of HSP70 against photoaging". Effect ”, Department of Medicinal Function Chemistry, Faculty of Pharmaceutical Education, Kumamoto University, Department of Medicinal Microbiology, 2011 Doctoral Dissertation).
(3) Summary As described above, the present inventors discovered that FADRCL contained a large amount of HSP70, HSP47, various paraclinic factors, etc. in Test 1, and in Test 5, FADRCL was excellent in these effects. It was discovered that it has a collagen-producing effect, and further, it was confirmed that HSP70 in FADRCL has a function of suppressing skin damage caused by ultraviolet rays (UVB), and the invention of use of the cosmetic of the present invention was completed.

<Discussion>
As described above, it has been demonstrated that the filtrate of the adipose tissue-derived stem cell disruption solution is extremely useful as a therapeutic agent for wounds on the skin and as a cosmetic. Using a filtrate of adipose tissue-derived stem cell disruption solution, which is a non-cell preparation, instead of adipose tissue-derived stem cells themselves, enables much higher efficacy and safer treatment than the previously reported treatment with stem cells or stem cell culture supernatants. To.

By using a skin protective agent containing the filtrate of the adipose tissue-derived stem cell disruption solution of the present invention as an active ingredient, it is possible to heal wounds on the skin more efficiently than before and reduce the risk of adverse events, and damage caused by ultraviolet rays. It can protect the skin from such things, and has great industrial applicability.

The present invention is not limited to the description of the embodiments and examples of the above invention. Various modifications are also included in the present invention to the extent that those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of the articles, published patent gazettes, patent gazettes, etc. specified in this specification shall be cited by reference in their entirety.

Claims (9)

1. A skin protective agent containing a filtrate of adipose tissue-derived stem cell disruption solution as an active ingredient, wherein the filtrate is one or more proteins selected from the group consisting of heat shock proteins, cytokines and / or paracrine factors and cell surface proteins. A skin protectant characterized by containing.
2. One or more proteins selected from the group consisting of heat shock proteins, cytokines and / or paracrine factors and cell surface proteins contained in the filtrate of the adipose tissue-derived stem cell disruption solution is a group consisting of HSP47, HSP70, HMGB1 and Annexin A6. The skin protective agent according to claim 1, which comprises one or more proteins selected from the above.
3. The skin protective agent according to claim 1 or 2, wherein the organism species of the adipose tissue-derived stem cells are hygiene.
4. The skin protectant according to any one of claims 1 to 3, wherein the skin protectant further contains any one or more of the following (a) to (c):
   (A) Adipose tissue-derived stem cells,
   (B) Crushed solution obtained by crushing adipose tissue-derived stem cells (a),
   (C) A supernatant obtained by centrifuging the crushed liquid (b).
5. A wound healing agent for promoting healing of a wound site of the skin, which comprises the skin protective agent according to any one of claims 1 to 4.
6. The wound therapeutic agent according to claim 5, wherein the wound is a trauma, an ischemic ulcer wound or a diabetic wound.
7. A cosmetic containing the skin protective agent according to any one of claims 1 to 4.
8. A method for producing a skin protective agent, which comprises the following steps (1) to (3):
   (1) A step of crushing the frozen stem cells derived from adipose tissue by freeze-thaw treatment.
   (2) The crushed solution obtained in step (1) or the supernatant obtained by centrifuging the crushed solution is filtered to obtain a filtrate, and (3) obtained in step (2). The step of formulating the filtrate.
9. The method for producing a skin protective agent according to claim 8, wherein instead of the step (1), a step of disrupting unfrozen adipose tissue-derived stem cells by ultrasonic treatment is included.

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