KR100614903B1 - Cosmetic Composition Containing Keratinocyte and/or Fibroblast - Google Patents

Abstract

The present invention comprises a cosmetic composition containing at least one of lyophilized keratinocytes and fibroblasts, and (1) culturing keratinocytes or fibroblasts; (2) lyophilizing the cultured keratinocytes or fibroblasts, and (3) adding at least one of the lyophilized keratinocytes and fibroblasts to a cosmetically acceptable carrier. It relates to a method for producing a cosmetic composition. Application of the cosmetic composition to the skin releases pre-expressed cytokines of keratinocytes and fibroblasts to promote skin regeneration.

Freeze drying, keratinocytes, keratinocytes, fibroblasts

Description

Cosmetic composition containing keratinocytes and / or fibroblasts {Cosmetic Composition Containing Keratinocyte and / or Fibroblast}

1A and 1B show the results of morphology of keratinocytes derived from skin tissue or hair follicles, respectively, using a phase contrast inverted microscope.

Figure 2 shows the results of observing the morphology of the fibroblasts derived from the skin tissue using a phase inverted microscope.

3A and 3B show the results of confirming the differentiation of keratinocytes derived from skin tissues or hair follicles through three-dimensional cultured skin models, respectively.

Figure 4 shows the pattern of the protein expressed in keratinocytes according to the culture period (lane 1: 8 days cultivation, lane 2: 9 days, lane 3: 10 days, lane 4: lyophilization after 10 days).

Figure 5 shows the pattern of the protein expressed in the fibroblasts according to the culture period (lane 1: serum, lane 2-5: protein pattern of the culture medium, 50, 70, 90, 100% confluency, respectively) Lanes 6-9: protein pattern of the extracted protein, 50, 70, 90, 100% confluency, lane 10: marker)

Figure 6 compares the amount of cytokines secreted by keratinocytes and fibroblasts before and after lyophilization.

7 shows the effects of keratinocytes (a) or fibroblasts (b) on the proliferation of keratinocytes through colony formation experiments.

Figure 8 shows the effect of keratinocytes or fibroblasts on the proliferation of fibroblasts.

9 is a graph showing the effect of keratinocytes or fibroblasts on collagen production of fibroblasts.

10 is a graph showing the stability of the cytokines of keratinocytes secreted from the composition containing keratinocytes.

Figure 11 shows the effect of the cosmetic composition containing keratinocytes and fibroblasts on the keratin moisture content.

Figure 12 shows the effect of the cosmetic composition containing keratinocytes and fibroblasts on the transdermal moisture evaporation.

The present invention relates to a cosmetic composition and a method for preparing the same, and a cosmetic composition containing at least one of lyophilized keratinocytes and fibroblasts, and (1) culturing keratinocytes or fibroblasts; (2) lyophilizing the cultured keratinocytes or fibroblasts, and (3) adding at least one of the lyophilized keratinocytes and fibroblasts to a cosmetically acceptable carrier. It relates to a method for producing a cosmetic composition.

The skin consists of the epidermis and the dermis, the outer layer of the epidermis consists of dead corneocytes, and the inner layer consists of living keratinocytes. Subdivided into four layers: the basal layer, the spinous layer, the granular layer and the cornified layer. Keratinocytes account for 90% of the epidermis, and the rest are melanocytes, T lymphocytes, Langerhans cells (macrophage), and Merkel cells, which are neurotransmitter-secreting cells. Actively proliferating keratinocytes are small cells (ie, basal cells) in contact with the basement membrane between the epidermis and the dermis, which grow in size when the differentiation begins, change the shape of the cytoplasm, Die while moving upstairs. The dermal layer below the epidermal layer is composed of fibroblasts and by-products thereof, and fibroblasts, a major cell group of the dermal layer, produce collagen and elastin, which serve as extracellular matrix. This fibroblast product is essential for the proliferation of keratinocytes in the epidermal layer. In addition to the epidermis and dermis described above, the skin includes hair follicles, sweat glands, sebaceous glands, and capillaries. There is keratinocytes in the hair follicles, and the interaction with adjacent fibroblasts induces the expression of morphogens and differentiates them into hairs.

In 1975, human keratinocyte culture was started by developing a culture method in Howard Green's laboratory at MIT University (Rheinwald and Green, Cell, 1975, vol. 6, pp. 331-344). It has also been applied to epithelial cells of. In addition, in 1979, keratinocytes were separated from a small amount of skin tissue and cultured for about two weeks to produce a sheet suitable for skin transplantation, followed by culturing keratinocytes for cultured skin fragments (Cultured Epidermal Graft, CEG). ) Was started. It was successfully applied to 95% burn surface area (BSA) children at Shriner's Hospital in Boston in 1984 and has been applied clinically in the United States and Europe (Gallico et al. New England Journal of Medicine, 1984, vol. 311). , pp. 448-451). However, self-derived cultured skin had a drawback of not being able to be applied directly to the wound because it took more than three weeks to culture the skin tissue. To compensate for this, the late 1980s began using cultured skin derived from other human skin tissues. In the early days, allogeneic cultured skin was transplanted like porcelain and was present in the human body and was thought to proliferate. Later, allogeneic skin is a wound healing dressing material that stimulates keratinocytes in the affected area to promote proliferation and movement. It turned out.

The exact mechanism of action is not known, but the excellent wound healing effect of keratinocytes has been confirmed by several researchers. This effect was found to be due to the action of cytokines and extracellular matrix (ECM) derived from keratinocytes. Cytokines secreted from keratinocytes are involved in immune and inflammatory responses and serve to regulate cell proliferation and migration. Sinetokines and extracellular substrates that produce and secrete keratinocytes and affect wound healing include basic FGF, TGF-α, PDGF, TGF-β, TNF, IL-1, IL-6, IL-8, IL-10, GM-CSF, and the like (Nowinski et al. Journal of Investigative Dermatology, 2003, vol. 122, pp. 216-221; Uchi et al. Journal of Dermatological Science, 2000, vol. 24, pp. S29-S38; Hashimoto, Journal of Dermatological Science, 2003, vol. 24, pp. S46-S50).

In addition, a number of unknown factors are secreted to stimulate the fibroblasts of the dermis to stimulate collagen production, thus creating an environment in which wound re-epithelialization can occur from wound and skin organs (Garner, Plastic and Reconstructive). Surgery, 1998, vol. 102, pp. 135-139). In particular, IL-1a acts on fibroblasts and promotes the expression of a growth factor called KGF, which acts as a representative paracrine factor in fibroblasts and mediates the proliferation of keratinocytes (Chedid). et al. Journal of Biological Chemistry, 1994, vol. 269, pp. 10955-10757; Mass et al. Journal of Cell Science, 1995, vol. 112, pp. 1843-1853).

That is, cytokines secreted from keratinocytes not only act directly on wound healing, but also indirectly promote wound healing by inducing the release of specific cytokines from other types of skin cells (Nowinski et al. Journal of Investigative Dermatology, 2002, vol. 119, pp. 449-455). In addition, enzymes such as MMP secreted from keratinocytes are involved in collagen synthesis and degradation, which inhibits the formation of hypertrophic scars due to collagen oversynthesis through a mechanism that promotes collagen synthesis and decreases over time in the early stages of wound healing. (CcCawley et al. Current Opinion in Cell Biology, 2001, vol. 13, pp.534-540).

As described above, excellent wound healing and skin regeneration effects of proteins expressed and secreted from keratinocytes and fibroblasts have been known, and various pharmaceutical compositions and cosmetic compositions using the same have been developed.

Japanese Patent Laid-Open No. JP3068506 discloses stresses such as lack of UV, temperature changes, magnetic fields, chemicals, essential nutrients during the culturing of animal cells, preferably keratinocytes or fibroblasts extracted from the hair of human skin, hair or eyelashes. Disclosed is a cosmetic composition prepared by providing a factor and in response to collecting and producing substances produced and secreted by the cells into a cosmetically acceptable carrier.

International Publication No. WO0114527 relates to a composition containing a growth factor produced in cultured skin cells, in vitro culturing skin cells such as keratinocytes and fibroblasts, and expressing and Disclosed are methods for collecting secreted cytokines and adding them to pharmaceuticals or cosmetics.

However, when using a protein expressed and secreted in keratinocytes or fibroblasts, the protein must be separated from the medium. In addition, the isolated protein itself is not only difficult to store, but may also lose its inherent biological activity when put into a cosmetic composition or pharmaceutical composition, and thus must be subjected to a process for preventing this.

Accordingly, the present inventors can achieve the above-described effects by incorporating keratinocytes and fibroblasts themselves into a cosmetically acceptable carrier in order to provide a cosmetic composition that can exert a continuous skin regeneration effect in a simple process. It has been confirmed that the present invention has been completed.

In one aspect, the present invention provides a cosmetic composition containing at least one of lyophilized keratinocytes and fibroblasts.

In another aspect (1) culturing keratinocytes or fibroblasts; (2) lyophilizing the cultured keratinocytes or fibroblasts, and (3) adding at least one of the lyophilized keratinocytes and fibroblasts to a cosmetically acceptable carrier. It provides a method for producing a cosmetic composition.

The cosmetic composition according to the present invention serves to promote skin regeneration. The "skin regeneration" means to increase the elasticity of the skin, improve wrinkles, inhibit the progression of aging, etc. When applying the cosmetic composition of the present invention to the skin release of cytokines previously expressed in keratinocytes or fibroblasts Initiate proliferation of keratinocytes or fibroblasts and reduce the death of existing cells to increase skin elasticity and delay and prevent the formation of wrinkles to delay aging.

As such, cytokines produced in keratinocytes or fibroblasts to promote skin regeneration include basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and keratinocyte growth factor. (keratinocyte growth factor (KGF), transforming growth factor alpha (TGFα), transforming growth factor beta-1 (TGFβ), granulocyte colony stimulating factor (GCSF) , Insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF), IL-1, IL-6, IL-8 , A number of interleukins, including IL-11, and the like.

The keratinocytes, one component of the cosmetic composition according to the invention, use those derived from animals, preferably mammals, more preferably humans. Differentiated skin, skin appendages or embryonic stem cells are suitable as the tissue of origin of keratinocytes. If the tissue of origin is skin, it is preferred to use those derived from foreskin, armpits, buttocks, breasts, scalp, pubis or scrotum. If the tissue of origin is a skin appendage, it is suitable to use one derived from hair follicles, sweat glands, sebaceous glands or capillaries. Hair follicles are preferably derived from hair follicles of anagen and are preferably used with hairs attached to keratinocytes of hair follicles.

The keratinocytes in the cosmetic composition of the present invention is included in an amount of 0.01% to 2.0% by weight, preferably 0.1% to 1.0% by weight, more preferably 0.1% to 0.2% by weight, based on the total weight of the composition.

Fibroblasts, which are other components of the cosmetic composition according to the present invention, use those derived from animals, preferably mammals, more preferably humans. The fibroblasts can be obtained by disaggregating differentiated skin tissue or by differentiating embryonic stem cells. Degradation may use methods known in the art, including methods of physical degradation and / or treatment of digestive enzymes and / or chelating agents that weaken connections with neighboring cells. This method can be used to disperse tissue into single cells without destroying the cells. In particular, separation by enzymes can be readily accomplished by first grinding the tissue and then treating any one of a number of digestive enzymes alone or in combination. Examples of suitable enzymes include but are not limited to trypsin, chymotrypsin, collagenase, elastase, hyaluronidase, DNase, pronase and dispase and the like. Physical decomposition methods include a grinder, a blender, a sieve, a homogenizer, or a sonicator.

In the cosmetic composition of the present invention, the fibroblasts are included in an amount of 0.01% to 5.0% by weight, preferably 0.05 to 2.0% by weight, more preferably 0.1 to 1.0% by weight, based on the total weight of the composition.

On the other hand, the cosmetic composition according to the present invention comprises the steps of (1) culturing keratinocytes or fibroblasts; (2) lyophilizing the cultured keratinocytes or fibroblasts, and (3) at least one of the lyophilized keratinocytes and fibroblasts may be added to a cosmetically acceptable carrier.

Keratinocytes or fibroblasts can be grown by culturing in conventional animal cell culture media. Such media may be prepared or commercially available, according to components and compositions known in the art. Medium that can be used in the present invention includes Dulbecco's Modified Eagle's Medium (DMEM); Minimal Essential Medium (MEM); M199; RPMI 1640; Iscove's Modified Dulbecco's Medium (EDMEM), MCDB, Ham's F-12, Ham's F-10, NCTC 109, NCTC 135, Keratinocyte-Serum Free Medium (keratinocyte-SFM), Keratinocyte Growth Medium (KGM) It doesn't happen. In the present invention, it is preferable to use Dulbecco's Modified Eagle's Medium (DMEM), Ham's F-12 or a mixture thereof for culturing keratinocytes, and Ham's F-12 or DMEM for culturing fibroblasts.

The medium is fetal bovine serum, bovine serum, triiodothyronine (T3), insulin, insulin, hydrocortisone, glutamine, adenine , Antibiotics such as transferrin, gentamycin, penicillin-streptomycin, and the like, may be further included, but are not limited thereto.

In the present invention, keratinocytes or fibroblasts are preferably lyophilized to increase stability at room temperature, reduce the need for costly cold storage, and to extend shelf life. The lyophilization process may consist of successive stages of freezing, primary drying and secondary drying. The primary drying step after freezing the composition is to drop the pressure and heat it for sublimation of water vapor. The secondary drying step is to evaporate residual moisture absorbed from the primary dry matter.

The keratinocytes or fibroblasts can be lyophilized according to lyophilization methods known in the art using commercially available lyophilizers. Such a method is disclosed, for example, in US Pat. No. 4,880,835 to Janoff et al. Prior to lyophilization, a lyophilized protective agent may be added to the dissolved cell solution. Lyophilized protectors protect cells and biological molecules so that their size and shape remain constant during the drying and thawing process. Lyophilized protective agents available are saccharides including dextrose, sucrose, maltose, mannose, galactose, raffinose, trehalose, lactose and trios. They are preferably added in an amount of 5 to 20% by weight, preferably about 10% by weight, to the solution in which the cells are lysed. In addition, an excipient may be added to the solution in which the cells are dissolved before the lyophilization process. Excipients include but are not limited to buffers of about 0.9% NaCl and about 10 mM sodium phosphate (pH 7.0M) or about 10 mM sodium citrate (pH 7.0).

In one embodiment, the method of lyophilizing keratinocytes or fibroblasts in the present invention is as follows: (1) Put the cells in a suitable container, float with buffer solution, and place in a cryogenic freezer at -70 to -80 ° C. Freeze for at least about 12 hours; (2) put the container into the sample container of the lyophilizer and slowly lower the pressure until the sample container pressure is about 1 × 10 ⁻³ torr; (3) the lyophilizer chamber temperature is maintained at about −50 ° C. and the sample container pressure is adjusted to about 2 × 10 ⁻⁴ torr and complete drying while maintaining the temperature and pressure for at least about 12 hours; (4) The container is taken out of the freeze dryer, sealed with a stopper, and stored at 2 to 8 ° C.

At least one of the lyophilized keratinocytes and fibroblasts may be added to a cosmetically acceptable carrier to provide a cosmetic composition according to the present invention. At least one of keratinocytes and fibroblasts in the cosmetic composition may be included as lyophilized itself or processed into microcapsules, particulates or lipid vesicles according to techniques commonly used in the art. It may also be formulated for use in admixture prior to application to any cosmetic composition by providing it in a physically isolated form (ie, stored in a separate container) rather than mixing with a cosmetically acceptable carrier.

Cosmetic compositions according to the present invention can be formulated into formulations of common cosmetic compositions (e.g., lotions, lotions, creams, gels, etc.) using techniques known in the art. The type and concentration of carrier that can be used depends on the desired formulation, but those skilled in the art can easily determine.

“Cosmetically acceptable carriers” include purified water, oils, waxes, fatty acids, fatty acid alcohols, fatty acid esters, surfactants, humectants, thickening agents, antioxidants, viscosity stabilizers. stabilizers), chelating agents, buffers, preservatives, lower alcohols, and the like. If necessary, a whitening agent, a moisturizer, an anti-inflammatory agent, an antibacterial agent, an antifungal agent, a vitamin, a sunscreen agent, an antibiotic, an anti-acne agent, a perfume, a dye may be included, and these are cosmetic compositions according to the present invention in amounts commonly used in the cosmetic field. Can be included. Suitable 0.01 to 20% by weight per total weight of the composition.

Specifically, hydrogenated vegetable oil, perm oil, cottonseed oil, olive oil, palm oil, jojoba oil, and avocado oil may be used as the oil, and waxes, wax, carnauba, candelilla, montan, ceresin, liquid paraffin, and lanolin may be used. This can be used. Stearic acid, linoleic acid, linolenic acid, oleic acid may be used as fatty acids, and cetyl alcohol, octyl dodecanol, oleyl alcohol, pantenol, lanolin alcohol, stearyl alcohol, hexadecanol may be used as fatty acid alcohol. Isopropyl myristate, isopropyl palmitate, butyl stearate may be used as the ester, but is not limited thereto.

Examples of the surfactant include anionic surfactants such as sodium stearate, sodium cetyl sulfate, polyoxyethylene laurylether phosphate, sodium N-acyl glutamate; Cationic surfactants such as stearyldimethylbenzylammonium chloride and stearyltrimethylammonium chloride; Amphoteric surfactants such as alkylaminoethylglycine hydrochloride and lecithin; Glycerin monostearate, sorbitan monostearate, sucrose fatty acid ester, propylene glycol monostearate, polyoxyethylene oleyl ether, polyethylene glycol monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene coconut fatty acid monoethanol Nonionic surfactants such as monoethanolarnide, polyoxypropylene glycol, polyoxyethylene castor oil, polyoxyethylene lanolin, and the like.

Glycerin, 1,3-butylene glycol, propylene glycol may be used as the hygroscopic agent, and ethanol or isopropanol may be used as the lower alcohol. Examples of thickeners include sodium alginate, sodium caseate, gelatin agar, xanthan gum, starch, cellulose ethers (e.g. hydroxyethyl cellulose, methyl cellulose, carboxymethyl cellulose, hydroxy propylmethyl cellulose), polyvinylpyrrolidone, Polyvinyl alcohol, polyethylene glycol, sodium carboxymethyl cellulose, and the like, but are not limited thereto. As antioxidants, butylated hydroxytoluene, butylated hydroxyanisole, propyl gallate, citric acid, ethoxyquin can be used, and chelating agents include disodium edetate and ethane hydroxy diphosphate. Citric acid, sodium citrate, boric acid, borax, disodium hydrogen phosphate are available as buffers, and methyl parahydroxybenzoate, ethyl parahydroxybenzoate, dihydroacetic acid as preservatives. Salicylic acid, benzoic acid may be used, but is not limited thereto.

Hereinafter, the examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, those skilled in the art. Will be self-evident.

<Example 1>

Culture of keratinocytes / fibroblasts derived from skin or hair follicles

(a) Cultivation of keratinocytes

Keratinocytes derived from skin or hair follicles were cultured using a 3: 1 mixture of gamma-irradiated 3T3 feeder, Dulbecco's modified Eagle medium and Ham-F12. After about 1cm ² skin tissue was treated with trypsin and single cellized, about 1 million skin cells were obtained and subcultured. In the case of hair follicles, outer roots of 20 hairs (ORS) were cultured. After 7 days more than 10,000 keratinocytes were obtained. At this time, 2 and 5 days after the inoculation, the cell growth patterns of keratinocytes were observed at a magnification of 100 times using a phase contrast inverted microscope (FIGS. 1A and B).

As shown in FIGS. 1a and b, the form in which keratinocytes form and grow colonies was similar regardless of the tissue from which keratinocytes were derived.

(b) Culture of fibroblasts

Fibroblasts are cultured based on Ham-F12 without single feeder after skin cells are inoculated at a density of 5 × 10 ⁵ in 162 cm ² culture vessels at 37 ° C and 10% CO ₂ . Cultured under conditions. This condition was used to selectively culture fibroblasts among the cells present in skin tissue. After 7 days, the selectively cultured fibroblasts were passaged by single cellized trypsin: EDTA. In the same manner as in Example 1 (a), after 2 and 5 days after inoculation, the cell growth pattern was observed at a magnification of 100 times using a phase contrast inverted microscope (FIG. 2).

As shown in FIG. 2, 5 days after the inoculation, the cells grew while maintaining the radial morphology of the fibroblasts to reach 100% confluency, and at this point, 4-5 × 10 ⁶ fibroblasts were obtained from the culture vessel.

(c) Lyophilization of keratinocytes and fibroblasts

When the cells obtained in (a) and (b) of Example 1 were cultured to reach 90% confluency, single cells were cultured using trypsin: EDTA to obtain keratinocytes and fibroblasts. It was.

The cells thus obtained were placed in a 50 ml tube and dispersed with about 5 ml of phosphate buffer solution, and the tube was placed in an cryogenic freezer and completely frozen at −75 ° C. for at least 12 hours. The tube was placed in a sample container of a pre-run lyophilizer and slowly depressurized until the sample container had a pressure of 1 × 10 ⁻³ torr. The lyophilizer chamber temperature was maintained at about −50 ° C. and the sample container pressure was adjusted to 2 × 10 ⁻⁴ torr. The temperature and pressure were maintained for at least 12 hours to complete drying. The tube was removed from the lyophilizer and sealed with a stopper and stored at 2-8 ° C.

Lyophilized cells were included in PBS or cosmetic compositions and used for cell proliferation analysis, ELISA analysis, protein pattern and content analysis according to the following examples.

<Example 2>

Comparison of differentiation ability of keratinocytes

Inoculating the keratinocytes obtained from Example 1 (a) on the collagen layer containing the fibroblasts obtained from Example 1 (b), using the same medium as in Example 1, 37 ° C., 10% CO ₂ , 80- Differentiation of skin tissue-derived keratinocytes and hair follicle-derived keratinocytes was induced by exposure to air after incubation for 5 days under conditions of 100% humidity. In other words, in order to float keratinocytes, which have reached 100% confluency on the collagen layer in the culture, in the air, an absorbent cotton pad is laid under the collagen layer so that the water level of the medium matches the paper. It was. In this state, the cultured and differentiated skin model was fixed with 10% formalin for 3 weeks, paraffin tissue was made, and then cut and subjected to H & E (Hematoxylin & Eosin) staining (FIGS. 3A and B).

As shown in Figure 3a and b was confirmed that the keratinocytes obtained in Example 1 form a base layer, a pole layer, a granule layer, a stratum corneum and normally differentiate regardless of the tissue derived.

<Example 3>

Comparison of protein expression pattern according to culture period of keratinocytes

0.2 × 10 ⁶ keratinocytes were inoculated onto the feeder and incubated for 8 days, 9 days and 10 days, and some of the cells cultured for 10 days were lyophilized. Cells were single cellized using trypsin: EDTA, and the protein was extracted by treating the single cell suspension with lysis buffer. The extracted protein was boiled in a sample buffer containing SDS (Sodium Dodecyl Sulphate), boiled, separated by size by 10% SDS-PAGE, and then visually evaluated by Coomassie blue staining. (FIG. 4).

As shown in FIG. 4, the protein expression pattern and the total expression amount of keratinocytes were constant regardless of the culture period, and there was no difference even when comparing the before and after lyophilization (lanes 3 and 4). From this it can be seen that lyophilization does not affect the protein expression and secretion of cells.

<Example 4>

Comparison of Protein Expression Patterns According to the Culture Period of Fibroblasts

Skin-derived fibroblasts were cultured to isolate cells, extract proteins and analyze protein expression patterns using SDS-PAGE as in Example 3 above (lanes 2 to 6 in FIG. 5). When the fibroblast confluency reached 50%, 70%, 90%, 100%, a portion of the culture medium was collected and analyzed for protein expression patterns using SDS-PAGE. 9).

As shown in Figure 5, the extracted protein pattern was constant regardless of time, but different from the protein pattern of the culture medium for analyzing the pattern of the secreted protein. Both cases showed a different pattern from the protein secreted from keratinocytes. The protein pattern of the culture medium was difficult to measure the change in protein pattern, as can be seen from the analysis of only 10% serum (lane 1 of Fig. 5), but the molecular weight 120 as the confluence increases The band (2 lines) was thickened between -200kD. It is assumed to be collagen because it is similar to the protein mass of collagen. On the other hand, the pattern of the extracted protein excluding the serum contained in the medium was found to increase the amount of all proteins including collagen as the confluence increases.

Example 5

Comparison of the amount of cytokines secreted by keratinocytes and fibroblasts

The amount of cytokines secreted from keratinocytes and fibroblasts before and after lyophilization was analyzed by ELISA. Specifically, the amount of IL-1α, bFGF, TGF-β, and PDGF secreted from cells was measured using the Quantikine Immunoassay Kit of the R & D System (FIG. 6).

As shown in FIG. 6, lyophilization did not affect the secretion of cytokines, and thus, cells killed by lyophilization also secreted a similar amount of cytokines as compared to living cells before lyophilization.

<Example 6>

Comparison of Colony Formability of Keratinocytes

TG004, a primary cultured keratinocyte, was inoculated about 100 on 3T3 feeder and incubated for 12 days. Lyophilized keratinocytes or fibroblasts were suspended in the keratinocyte culture solution at a concentration of 0.5 × 10 ⁶ / ml and used as a culture medium. After the incubation, 10% formalin solution was added to fix and stained with Rhodamine blue solution. Colony Forming Efficiency (CFE) was defined as the ratio of the number of colony forming to the number of cells inoculated.

[Equation 1]

% Colony forming capacity (CFE) = number of colony forming cells / inoculated cells × 100

As shown in FIG. 7, when the culture medium without lyophilized cells was used (control), the colony forming ability of the cells was 35%, and the culture medium containing keratinocytes or fibroblasts was 46% and 44%, respectively. Colony forming ability was shown. In particular, the colony size increased from 3.2 mm in diameter to 8.3 mm and 7.9 mm, respectively. From this, keratinocytes or fibroblasts containing cultures secrete cytokines necessary for the proliferation and migration of keratinocytes, inducing an increase in colony number and size.

<Example 7>

Comparison of Proliferative Capacity of Fibroblasts

0.5 x 10 ⁵ fibroblasts were cultured in a 24-well plate using DMEM containing 10% fetal bovine serum. After 24 hours, lyophilized keratinocytes or fibroblasts were suspended in the culture medium at a concentration of 0.5 × 10 ⁵ / ml while replacing with DMEM containing 5% fetal bovine serum. After culturing for 24 hours, the growth rate of fibroblasts was measured by the method of tetrazolium salt 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide (MTT). MTT solution (5mg / ml in PBS) was added to the plate and reacted for 4 hours under humidification condition of 37 ° C. After removing the culture solution, a lysis solution consisting of 10% SDS and 45% dimethyl foramide at pH4.7 The reaction was carried out for 12 to 24 hours after the addition of M. Fortraon (3- (4,5-dimethylthiazol-2-yl) -2,5- The ability of mitochondria to reduce to diphenyl-tetrazolium bromide), ie, cell proliferation, was determined by measuring absorbance at 590 nm (FIG. 8).

As shown in FIG. 8, when the fibroblasts were cultured using a culture solution containing keratinocytes or fibroblasts, the growth rate was increased by 21-30%. Therefore, it can be seen that keratinocytes or fibroblasts promote the proliferation of fibroblasts as well as the proliferation of keratinocytes as in Example 6.

<Example 8>

Comparison of Collagen Production Capacity of Fibroblasts

It was evaluated whether keratinocytes or fibroblasts contained in the cosmetic composition can promote skin regeneration by measuring collagen, which is one of the extracellular matrix expressed by the fibroblasts, which are indicator cells. After culturing under the same conditions as in Example 7 and mixing 500 μl of conditioned medium and 1 ml of Sirius Red staining reagent collected while culturing, absorbance was measured at 540 nm (FIG. 9).

As shown in FIG. 9, 35.4% and 25.6% of the culture medium containing keratinocytes or fibroblasts was used as compared to the case of culture using the keratinocytes or the fibroblast-free culture medium (control). Collagen production increased.

Example 9

Stability of Compositions Containing Keratinocytes and Fibroblasts

By measuring the amount of IL-1α, which is one of the factors secreted from keratinocytes, as in Example 5, it is evaluated whether keratinocytes in the composition can stably secrete proteins and exhibit skin regeneration efficacy over time. It was. This may provide a criterion to determine how long the efficacy of the composition containing the cells is maintained (FIG. 10).

As shown in Fig. 10, the secretion amount of IL-1α in the composition containing keratinocytes was maintained at a stable level showing more than 70% of the secretion amount during preparation even after 8 weeks.

<Example 10>

Preparation of cosmetic composition containing keratinocytes and fibroblasts

As an essence containing the lyophilized keratinocytes and fibroblasts obtained in order to apply the cosmetic composition according to the present invention directly to human skin and confirm the activity and stability of cytokines secreted from the cells contained in the formulation. Formulated.

Table 1

Cosmetic composition containing keratinocytes and fibroblasts

Ingredient group Ingredient number ingredient Component ratio of composition (A) Component ratio of composition (B) A One Purified water Add up to 100g total Add up to 100g total 2 1,3 beta glucan 5.0 5.0 3 Pentylene glycol 3.0 3.0 4 Sodium hyaluronate 0.15 0.15 5 Citric acid 1.44 1.44 6 glycerin 3.0 3.0 B 7 Carbomer 0.14 0.14 C 8 ethanol 3.0 3.0 9 POE 60 Cured Castor Oil 0.5 0.5 10 Methylparaben 0.1 0.1 D 11 Sodium hydroxide 1.0 1.0 E 12 Lyophilized keratinocytes and fibroblasts 0 0.2

Among the components in the table, the ingredients of group A were added to the container, mixed using a mixer to form an aqueous solution phase, and then the ingredients of group B were slowly added to the aqueous solution to be sufficiently stirred to be homogeneous. The components of the C group were slightly warmed (50 degrees) and homogenized in a separate container, and then stirred and slowly added to the mixed solution of the A and B group components to form micelles. Component D was then neutralized to impart viscosity, and in the case of composition (B), component E was added to prepare a slime cosmetic.

<Example 11>

Skin regeneration experiment of cosmetic composition including keratinocytes and fibroblasts

For volunteers with atopic dermatitis, the composition (A) or composition (B) prepared in Example 10 was applied for 4 weeks to determine how quickly the skin was regenerated by measuring the stratum corneum moisture retention and transdermal skin moisture content.

Thirty volunteers (hereinafter referred to as subjects) who were diagnosed as atopic patients with mild to severe atopic dermatitis (ie, mild atopy evenly spread throughout the skin) were selected. Prior to the application of the cosmetic composition according to the present invention, the skin of the subject was photographed, skin hydration and skin moisture loss (TEWL) were measured, and the external symptoms of the skin were recorded. From these results, two sites (hereinafter, referred to as measurement sites) in which each of the subjects had a similar value within the error range and the amount of water loss and skin moisture were selected. The subject then directly applied composition (A) or composition (B) twice daily (morning and evening) to the measurement site.

The stratum corneum moisture content and transdermal moisture evaporation were measured once a week for 6 weeks (prior to application, 1 week, 2 weeks, 4 weeks, 6 weeks (total 5 measurements). After adjusting for 30 minutes or more in a constant temperature and humidity room (20 ℃, 50% humidity) was measured.

(a) Measurement of moisture content in the stratum corneum

Moisture content in the stratum corneum of the measurement site was measured using a moisture meter (corneometer, C & K) (unit: corneometer units) and the measured values are shown in Table 2 and FIG. 11.

Table 2

Results on stratum corneum retention

Composition (A) Composition (B)  visit Average Standard Deviation Average Standard Deviation Week 0 31.21 14.71 44.67 11.59 1 week 38.58 15.36 49.96 17.28 2 weeks 37.19 17.40 49.30 13.37 4 Weeks 36.61 14.76 47.51 16.33 6 Weeks 38.54 11.99 49.04 15.71

Looking at the water content of the stratum corneum between the compositions it can be confirmed that the water content of the measurement site to which the composition (B) is applied than the composition (A). In each case, it was found that the water content increased with time. The repeated measurement analysis showed that there was a significant difference in the moisture content of the stratum corneum between the composition (A) and the composition (B) (p value = 0.011). There was no significant difference between my time points (p value = 0.09664). Although not statistically significant, the moisture content increased with time compared to 0 weeks and the moisture content of 6 weeks increased by 23.5% compared to 0 weeks.

(b) transdermal moisture evaporation measurement

The transdermal moisture evaporation amount of the measurement site was measured using a moisture evaporation meter (tewameter) (unit: g / m ² / h) and the measured values are shown in Table 3 and FIG. 12.

Table 3

Results for transdermal moisture evaporation

Composition (A) Composition (B) visit Average Standard Deviation Average Standard Deviation Week 0 21.16 13.69 8.14 10.60 1 week 25.09 18.74 10.17 7.18 2 weeks 24.56 19.35 7.75 5.61 4 Weeks 20.43 15.42 6.13 4.99 6 Weeks 18.32 13.65 6.91 4.01

Looking at the transdermal moisture evaporation between the compositions it was confirmed that the composition (A) is higher than the composition (B). In the case of the composition (B) it can be seen that from 1 week at 8.14 to 0 weeks and then slightly decreased from 2 weeks. In the case of composition (A), the amount of water evaporation increased from 21.16 at 0 weeks to 25.09 at 1 weeks, and from then on, the amount of water evaporated gradually decreased.

Repeated analysis showed a significant difference in transdermal water evaporation between composition (A) and composition (B) (p value = 0.0055), but no significant difference between time points in each composition (p value = 0.4373). Although not statistically significant, water evaporation at 6 weeks decreased by about 13.4% compared to water evaporation at 0 weeks.

It was confirmed through the above example that lyophilized keratinocytes and fibroblasts can induce rapid skin regeneration even in the form contained in the cosmetic carrier.

Cosmetic compositions containing any of the fibroblasts of lyophilized keratinocytes can exert a sustained skin regeneration effect when applied to the skin by secreting previously expressed cytokines over a long period of time.

Claims (10)

Cosmetic composition comprising at least one of keratinocytes and fibroblasts as a cell in a lyophilized state. According to claim 1, wherein the keratinocytes and fibroblasts are basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), keratinocyte growth factor (KGF), transforming growth factor alpha (TGFα), transfection Secretes cytokines selected from the group consisting of transforming growth factor beta (TGFβ), granulocyte formation stimulating factor (GCSF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF) and interleukin Cosmetic composition, characterized in that. According to claim 1, wherein the keratinocyte cosmetic composition, characterized in that it comprises 0.01 to 2.0% by weight based on the total weight of the cosmetic composition. The cosmetic composition of claim 1, wherein the keratinocytes are derived from differentiated skin, skin appendages, or embryonic stem cells. The cosmetic composition of claim 4 wherein the skin is selected from the group consisting of foreskin, armpits, buttocks, breasts, scalp, pubis and scrotum. The cosmetic composition according to claim 4, wherein the skin appendage is selected from the group consisting of hair follicles, sweat glands, sebaceous glands and capillaries. According to claim 1, wherein the fibroblast is a cosmetic composition, characterized in that it comprises 0.01 to 5.0% by weight per weight of the cosmetic composition. The cosmetic composition according to claim 1, wherein the fibroblasts are derived from differentiated skin or embryonic stem cells. The cosmetic composition of claim 1 wherein the cosmetic composition is formulated as a lotion, lotion, cream or gel. (1) culturing keratinocytes or fibroblasts; (2) lyophilizing the cultured keratinocytes or fibroblasts, and (3) adding at least one of the lyophilized keratinocytes and fibroblasts to a cosmetically acceptable carrier. Method for producing a cosmetic composition according to any one of claims 1 to 8.

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