WO2006014089A1

WO2006014089A1 - Cosmetic composition containing keratinocyte and/or fibroblast

Info

Publication number: WO2006014089A1
Application number: PCT/KR2005/002555
Authority: WO
Inventors: Sae-Wha Jeon; Dong-Il Jang
Original assignee: Tego Science Inc.
Priority date: 2004-08-06
Filing date: 2005-08-05
Publication date: 2006-02-09
Also published as: KR20060013207A; JP2006045175A; KR100614903B1

Abstract

Disclosed herein is a cosmetic composition comprising lyophilized keratinocytes and/or fibroblasts and a method of preparing the cosmetic composition, comprising (1) culturing keratinocytes or fibroblasts; (2) lyophilizing the cultured keratinocytes or fibroblasts; and (3) incorporating the lyophilized keratinocytes and/or fibroblasts into a cosmetically acceptable carrier. When applied to the skin, the keratinocytes and/or fibroblasts contained in the cosmetic composition release already expressed cytokines to promote skin regeneration.

Description

COSMEπC COMPOSITION CONTAINING KERATINOCYTE AND/OR FIBROBLAST

TECHNICAL FIELD

The present invention relates to a cosmetic composition and a method of preparing the same.

More particularly, the present invention relates to a cosmetic composition containing fieeze-dried keratinocytes and/or fibroblasts and a method of preparing the same, comprising (1) culturing keratinocytes or fibroblasts; (2) fieeze-drying the cultured keratinocytes or fibroblasts and (3) incorporating freeze-dried keratinocytes or fibroblasts into cosmetically acceptable carriers.

BACKGROUND ART

The skin consists of two layers: the epidermis and the dermis (coriurn). Largely composed of the outer homy corneocyte layer and the inner live keratinocyte layer, the epidermis has four morphologically distinct layers which are, from the innermost layer out, a basal layer, a spinous layer, a granular layer and a cornified layer. Keratinocytes constitute about 90% of the epidermis, with melanocytes, T lymphocytes, Langerhans cells, neurotransrnitter-secreting Merkel cells, etc. Actively growing keratinocytes are small cells (basal cells) in contact with the basement membrane between the epidermis and the dermis. When they start to differentiate, keratinocytes become large in size and have morphologically changed cytoplasms, migrating to the outer layers of the epidermis where apoptosis takes place, hi the dermis beneath the epidermis, fibroblasts and their by-products are found. Fibroblasts, predominant in the dermis, are responsible for the production of extracellular matrices such as collagen and elastin, which are essential for the proliferation of keratinocytes in the epidermis. In the dermis, the skin appendages, such as hair follicles, sweat glands and sebaceous glands,=are found. Li hair follicles, keratinocytes interact with adjacent fibroblasts, which induces the expression of morphogen leading to differentiation to hair.

In 1975, Dr. Howard Green's laboratory of MTT, U.S.A., developed a method of culturing human keratinocytes (Rheinwald and Green, Cell, 1975, vol.6, pp.331-344), and successfully applied the method to other types of epithelial cells. In 1979, they started to prepare cultured epidermal grafts (CEGs) transplantable to humans, by successfully separating keratinocytes from skin tissue, culturing the cells for about two weeks to prepare sheets suitable for transplantation. Since 1986, when CEGs were successfully transplanted onto a boy with 95% BSA (Bum Surface Area) in Shriner's Hospital at Boston, CEGs have been clinically applied in US and Europe (Gallico et al. New England Journal of Medicine, 1984, vol.311, pp.448-451). The autologous CEGs₅ however, present a disadvantage in terms of time required to culture cells to the number sufficient enough to cover the body of a patient To overcome this disadvantage, allogeneic cultured epidermal grafts (derived from keratinocytes of other persons) have been used since the late 1980's. At first, allogeneic grafts were believed to be taken at the transplanted site, as in the case of autologous grafts. But, allogeneic cultured grafts were later proven to act as a biological dressing material for wound healing by stimulating remaining keratinocytes within wounds to proliferate and migrate.

Although their exact mechanism of action remains to be proven, keratinocytes have been reported to exert outstanding wound healing effects in many studies. These wound healing effects are attributable to the action of the cytokines and extracellular matrix (ECM) derived from keratinocytes. Cytokines secreted from keratinocytes and involved in immune and inflammatory responses play an important role in cell proliferation and migration. The cytokines and growth factors secreted by keratinocytes and involved in wound healing include basic FGF, TGF-α, PDGF, TGF-β, TNIF, IL-I, IL-6, IL-8, IL-10, GM-CSF, etc. (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, keratinocytes secrete various unknown factors in combination, which act to stimulate dermal fibroblasts to produce collagen, thereby creating an environment in which re- epiftielialization occurs from wound surroundings and skin appendages (Garner, Plastic and Reconstructive Surgery, 1998, vol.l02,pp.l35-139). Particularly, IL-lα induces fibroblasts to express KGF, which acts as a paracrine factor to mediate tfis proliferation of keratinocytes (Chedid et al. Journal of Biological Chemistry, 1994, vol.269, pp.10753-10757; Mass et al. Journal of Cell Science, 1995, vol.H2,pp.l843-1853).

Accordingly, Hie cytokines secreted from keratinocytes not only act directly to close wounds, but are also indirectly involved in wound healing by inducing skin cells of different types to secrete certain cytokines (NowinsM et al. Journal of Investigative Dermatology, 2002, vol.119, pp.449- 455). Furthermore, MMP, a keratinocyte-secreted enzyme responsible for collagen synthesis and degradation, promotes collagen synthesis in the early stages of a wound healing process and degrades collagen in the late stages so as to prevent hypertrophic scar formation attributed to the overproduction of collagen (McCawley et al. Current Opinion in Cell Biology, 2001, vol.13, pp.534-540).

With an increase in information on the effects of proteins expressed or secreted from keratinocytes and fibroblasts on wound healing, a variety of pharmaceutical and cosmetic compositions including these proteins have been developed.

Japanese Pat Laid-Open Publication No. 3068506 discloses a cosmetic composition comprising the substances which keratinocytes or fibroblasts, derived from animal tissues, preferably human skin, hair or hair shafts of eyebrows, produce or secrete in response to stresses such as those induced by UV irradiation, temperature change, magnetic fields, chemicals, deficiency of essential nutrients, etc., and cosmetically acceptable carriers.

WOOl 14527 describes compositions containing growth fectors synthesized from cultured skin cells. This patent discloses a method in which cytokines secreted into the culture medium from cultured keratinocytes and fibroblasts are collected and incorporated into pharmaceutical or cosmetic preparations. In utilizing the proteins synthesized or secreted by keratinocytes or fibroblasts, a process for separating the proteins from the culture medium must be pursued, likely leading to the loss of unknown functional factors. In addition, because the proteins, once separated, are themselves not only difficult to store, but may also lose their intrinsic biological activities when incorporated into cosmetic or pharmaceutical compositions, they must undergo aprocess to prevent these problems.

DISCLOSURE OF INVENTION

Leading to the present invention, an intensive and thorough research, conducted by the present inventors, on skin regeneration resulted in the finding that freeze-dried keratinocytes and/or fibroblasts incorporated with a cosmetically acceptable carrier can steadily release the cytokines responsible for skin regeneration.

In accordance with an aspect of the present invention, a cosmetic composition comprising at least either or both of lyophilized keratinocytes and lyophilized fibroblasts is provided In accordance with another aspect of the present invention, a method of preparing a cosmetic composition, comprising (1) cultaring keratinocytes or fibroblasts; (2) lyophffizing the cultured keratinocytes or fibroblasts and (3) incorporating the lyophilized keratinocytes or fibroblasts into a cosmetically acceptable carrier is provided.

BRIEF DESCRIPTION OF TBE DRAWINGS

FIGS. IA and IB are photographs, taken using a phase-contrast inverted microscope, showing tie morphologies of skin and hair-follicular keratinocytes respectively.

FIG. 2 is photographs, taken using a phase-contrast inverted microscope, showing the morphologies of dermal fibroblasts.

FIGS. 3A and 3B are microphotographs of from skin and hair-follicular keratinocytes, respectively, showing their differentiation in athree-dimensionally cultured skin model. FIG.4 shows patterns of the proteins expressed by keratinocytes cultured for 8 days (lane 1),

9 days (lane 2), 10 days (lane 3), and for 10 days and freeze-dried (lane 4).

FIG. 5 shows patterns of the proteins expressed by fibroblasts cultured for different periods (lane 1: serum, lanes 2-5: proteins secreted into culture medium at 50, 70, 90 and 100% confluency, respectively, lanes 6-9: proteins extracted from cells at 50, 70, 90, 100% confluency, respectively, lane 10: marker)

FIG. 6 is a bar graph showing amounts of cytokines secreted from keratinocytes and fibroblasts before and after lyophilization.

FIG. 7 is photographs of culture dishes in which keratinocyte colonies are formed, showing the effects of keratinocytes (a) and fibroblasts (b) on the proliferation of keratinocytes. FIG. 8 is a bar graph showing the effect of keratinocytes or fibroblasts on the proliferation of fibroblasts.

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

FIG. 10 is a line graph showing the steady secretion of cytokines from keratinocytes that are contained in a composition over time. FIG. 11 is a line graph showing the effect of a cosmetic composition comprising both keratinocytes and fibroblasts on skin hydration

HG. 12 is a line graph showing die effect of a cosmetic composition comprising both keratinocytes and fibroblasts on transepidermal water loss.

BEST MODES FOR CARRYING OUT THE INVENTION

The cosmetic composition in accordance with the present invention acts to promote skin regeneratioa The term "skin regeneration" as used herein means elasticity improvement, wrinkle reduction and aging retardation in the skin. If the cosmetic composition of the present invention is applied to the skin, the cytokines already secreted from keratinocytes and/or fibroblasts are released into the skin to promote the proliferation of keratinocytes or fibroblasts and reduce the death of preexisting cells, thereby retarding the aging process of skin through the increase of skin elasticity, and the prevention or retardation of skin wrinkle formation. The keratinocyte- or fibroblast-produced cytokines that may act to promote skin regeneration include basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), keratinocyte growth iactor (KGF), transforming growth iactor alpha (TGFα), transferming growth factor beta-1 (TGFβ), granulocyte colony stimulating factor (GCSF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), tumor necrosis iactor (TNF), interleukins such as IL-I, IL-6, EL-8, DL-Il, etc., but are not limited thereto.

Keratinocytes, a main constituent of the cosmetic composition according to the present invention, may be obtained from animals, preferably from mammals, and more preferably from humans. As for the tissues of origin for keratinocytes, differentiated skin, skin appendages, and embryonic stem cells are preferred Suitable as the skin origins are foreskin, scalp, armpits, hips, breasts, pubis, or scrotum. Preferable examples of the skin appendages from which keratinocytes can be obtained include hair follicles, sweat glands and sebaceous glands. As for hair follicles, it is preferred that keratinocytes are obtained from hair follicles of anagea

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

Fibroblasts, another or an alternative constituent of the cosmetic composition according to the present invention, may be taken from animals, preferably from mammal, and more preferably from humans. The disaggregation of differentiated skin tissues and/or the differentiation of embryonic stem cells can yield fibroblasts. Disaggregation is well known in the art and includes physical disaggregation and/or treatment with digestive enzymes and/or chelating agents that weaken the junction between adjacent cells. The disaggregation methods can disperse skin tissues to yield single cells without causing cell rupture. Particularly, enzymatic disaggregation can be readily achieved by mincing tissues and treating them with single or combinations of digestive enzymes. Examples of suitable enzymes include trypsin, chymotrypsin, collagenase, elastase, hyaluronidase, DNase, pronase, and dispase, but are not limited thereto. As for physical disaggregation, a grinder, a blender, a sieve, a homogenizer or a sonicator may be used

Fibroblasts are contained in an amount of 0.01 to 5.0 % by weight based on the total weight of the cosmetic composition of the present invention, preferably in an amount of 0.05 to 2.0 % by weight, and more particularly in an amount ofO.l to 1.0 %by weight The cosmetic composition according to the present invention can be prepared by (1) culturing keratinocytes or fibroblasts; (2) freeze-drying the cultured keratinocytes or fibroblasts; and (3) incorporating the freeze-dried keratinocytes or fibroblasts into a cosmetically acceptable carrier.

To obtain a desired amount of keratinocytes or fibroblasts conveniently, in vitiv culturing using conventional culture media for animal cells is preferred Cell culture media can be prepared according to methods that are well known in the art or are commercially available. Media suitable for the culture of keratinocytes and/or fibroblasts are exemplified by Dulbecco's Modified Eagle's Medium(DMEM); Minimal Essential Medium(MEM); M199; RPM 1640; kcove's Modified Dulbecco's Medium(EDMEM), MCDB, Ham's F-12, Ham's F-IO, NCTC 109, NCTC 135, keratinocyte-serum free medium (keratinocyte-SFM), and keratinocyte growth medium (KGM), but are not limited thereto. Preferably, keratinocytes are cultured in Dulbecco's Modified Eagle's Medium(DMEM), Ham's F-12 or mixtures thereof while either Ham's F-12 or DMEM is used to culture fibroblasts.

The media may be supplemented with fetal bovine serum, bovine serum, triiodothyronine (T3), insulin, hydrocortisone, glutamine, adenine and/or transferrin, and antibiotics such as gentamicin and penicillin-streptomycin may be added to the media.

In accordance with the present invention, a freeze-drying process for keratinocytes or fibroblasts is employed in order to improve stability at room temperature, to eliminate the necessity for high cost, low temperature storage, and to extend the shelf life thereof. The fieeze-drying process may be completed by sequentially conducting a freezing step, a primary drying step and a secondary drying step. In the primary drying step after the freezing step, moisture is sublimated at an elevated temperature under a reduced pressure. The remaining moisture is evaporated from the primarily dried cells in the second drying step.

Using a commercially available lyophilizer, keratinocytes or fibroblasts can be freeze-dried according to a method well known in the art, as disclosed, for example, in U. S. Pat No. 4,880,835, granted to Janoff et al. Preceding lyophilization, a cryoprotectant may be added to a cell suspension. The cryoprotectant helps protect what is being frozen from freezing damage, and serves to keep the sizes and morphologies of cells and biological molecules constant during the freezing and thawing processes. Suitable cryoprotectants are saccharides including dextrose, sucrose, maltose, mannose, galactose, kffinose, trehalose, lactose, and triose. They may be added to a cell suspension in an amount of about 5 to 20 % by weight and preferably in an amount of about 10 % by weight In addition, excipients may be added to the cell suspension before the lyophilization or freeze-drying process. The examples of available excipients are, but not limited to, buffers of approx. 0.9% NaCl, and approx. IDmM sodium phosphate (pH7.0) or approx. 1OmM sodium citrate (pH 7.0).

In an embodiment of the present invention, the lyophilization of keratinocytes or fibroblasts is conducted as follows: (1) cells are suspended in a buffer in a suitable vessel, followed by freezing the cell suspension at -70 to -80°C for about 12 hours or longer in a deep freezer; (2) the vessel is put in a sample container in a lyophilizer and the inner pressure of the sample container is slowly reduced to about 1x10^'3 torr; (3) while the lyophilizer chamber is maintained at about -50⁰C₉ the inner pressure of the sample container is further controlled to about 2xlO⁴ torr and kept thereat for 12 hours or longer to complete the lyophilization; and (4) the vessel is withdrawn from the lyophilizer, sealed with a cap, and stored at 2 to 8⁰C.

The freeze-dried keratinocytes and/or fibroblasts are combined with a cosmetically acceptable carrier to provide a cosmetic composition in accordance with the present invention. In the cosmetic composition, the freeze-dried keratinocytes and/or fibroblasts may be contained as they are or in the form of microcapsules, microparticles, or liposomes. Alternatively, the keratinocytes and/or fibroblasts may be formulated to be provided in a physically separated form (e.g., stored in a separate vessel) rather than incorporated into a cosmetically acceptable carrier, so as to be mixed with another cosmetic composition immediately prior to application to the skin.

Using well-known processes, the cosmetic composition according to the present invention may be prepared in general formulation forms (e.g., lotions, creams, gels, etc.). There are various kinds of available carriers, and those who are skilled in the art can readily determine the kinds of carriers and concentrations thereof suitable for desired purposes.

Illustrative, but non-limitative examples of the "cosmetically acceptable carrier" as used herein include purified water, oils, waxes, fatty acids, fatty acid alcohols, fatty acid esters, surfactants, humectants, thickening agents, antioxidants, viscosity stabilizers, chelating agents, buffers, preservatives, lower alcohols, etc. Other additives that may be added when necessary are exemplified by whiteners, moisturizers, anti-inflammatory agents, antibacterial agents, antifungal agents, vitamins, UV- blocking agents, antibiotics, anti-acne agents, perfumes, dyes, etc. Ih the cosmetic composition according to the present invention, the additives may be contained in amounts that are typically proposed in the cosmetic field. Generally, suitable amounts of the additives fell into the range from 0.01 to 20 % by weight of the composition.

Concrete examples of available additives include hydrogenated vegetable oils, castor oil, cotton seed oil, olive oil, coconut oil, jojoba oil, and avocado oil for oils; beeswax, spermaceti wax, carnauba wax, candellila wax, montan, ceresin, liquid paraffin, and lanolin for waxes; stearic acid, linoleic acid, linolenic acid, and oleic acid for fatty acids; cetyl alcohol, octyl dodecanol, oleyl alcohol, panthenol, lanolin alcohol, stearyl alcohol, and hexadecanol for fatty acid alcohols; and isopropyl myristearate, isopropyl palrnitate, and butyl stearate for fatty acid esters, but are not limited thereto.

As for surfactants, they may be anionic surfactants such as sodium stearate, sodium cetylsulfate, polyoxyethylene laurylether phosphate, sodium N-acyl glutamate; cationic surfactants, such as steaiylo^ethylbenzylammonium chloride and stearyltrimethylammonium chloride; amphophilic surfactants, such as alylaminoethylglycin hydrochloride and lecithin; or non-ionic surfactants, such as glycerin monostearate, sorbitan monostearate, sucrose fatty acid ester, propylene glycol monostearate, polyoxyethylene oleylether, polyethylene glycol monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene coconut fatty acid monoethanolarnide, polyoxypropylene glycol, polyoxyethylene castor oil, and polyoxyethylene lanolin.

Glycerin, 1,3-butylene glycol, and propylene glycol may be used as humectants. Efhanol and isopropyl alcohol may be used as suitable lower alcohols in the cosmetic composition of the present inventioa Examples of usable thickening agents include sodium alginate, sodium caseinate, gelatin, agar, xanthan gum, starch, cellulose ethers (e.g, hydroxyefhyl cellulose, methyl cellulose, carboxymethyl cellulose, hydroxyl propylmethyl cellulose), polyvinylpyrrolidone, polyvinylalcohol, polyethylene glycol, and sodium carboxymethyl cellulose, but are not limited thereto. Available antioxidants may be exemplified by butylated hydroxytoluene, burylated hydroxyanisole, propyl gallate, citric acid and ethoxyquin, usable chelating agents by disodium edetate and ethanehydroxy diphosphate, usable buffers by citric acid, sodium citrate, boric acid, borax, and disodium hydrogen phosphate, and available preservatives methyl parahydroxybenzoate, ethyl parahydroxybenzoate, dihydroacetic acid, salicylic acid and benzoic acid, but these are only illustrative, and not at all limitative.

EXAMPLES

EXAMPLE 1 : Culturing of Keratinocytes/Fibroblasts from Skin or Hair Follicles

Culturing of keratinocytes

Keratinocytes originating from the skin or hair follicles were cultured in a mixture of 3:1 Dulbecco's modified Eagle medium and Ham-F12, using a gamma ray-irradiated 3T3 feeder system. A 1 cm²-sized piece of skin tissue was treated with trypsin and a single cell suspension of one million keratinocytes were obtained and subcultured. For hair-follicular keratinocytes, the outer root sheaths (ORS) of 20 hairs were cultured for seven days to afford ten thousand or more keratinocytes. Two and five days after inoculation, keratinocytes were observed for growth morphology using a phase-contrast inverted microscope at a magnification of 100 (FIGS. IA and IB). As shown in FIGS. IA and IB, keratinizing colonies formed from single cells were found to be morphologically identical, regardless of their origins.

(b) Culturing of fibroblasts

After skin tissue was separated into single cells, they were inoculated at a density of 5x10⁵ cells into a Ham-F12 medium in a culture dish having an area of 162 cm², without using a feeder and were cultured at 37⁰C in a 10% CO² atmosphere. Under this condition, fibroblasts could be exclusively cultured out of skin cells. 7 days later, the selectively cultured fibroblasts were treated with trypsin:EDTA until they were separated into single cells, which were then subcultured. Two and five days after inoculation, as in (a) of Example 1, the dermal fibroblasts were observed for morphology using a phase-contrast inverted microscope at a magnification of 100 (FIG.2).

Five days after inoculation, as seen in FIG. 2, fibroblasts were grown at 100% confluency, maintaining characteristic radial spreading. At this time, 4~5xlO⁶ fibroblast cells were obtained from the culture dish.

(c) Lyophilization of keratinocytes and fibroblasts

The cells obtained in Examples l(a) and l(b) were cultured to 90% confluency, followed by treatment with trypsin:EDTA to separate keratinocytes and fibroblasts into single cells.

These cells were put in 50ml tubes and suspended in about 5ml of phosphate buffered saline, after which the tubes were completely frozen at -75°C for about 12 hours in a deep freezer. The tubes were placed in a sample container of a freeze-dryer that was already operating and the pressure of the container was slowly reduced to 1x10^'3 torr. While the chamber of the freeze-dryer was maintained at about -50⁰C, the pressure of the sample container was controlled to 2XlO^"4 torr. Maintaining of the temperature and pressure for 12 hours or longer led to complete lyophilization. The tubes were taken out of the freez-dryer, sealed with caps, and stored at 2 to 8°C. The lyophilized cells were contained in PBS or a cosmetic composition for use in analysis of cell proliferation, ELISA analysis, and analysis for protein pattern and content.

EXAMPLE 2: Comparison of Capacity to Differentiate

On a collagen layer containing the fibroblasts obtained in Example l(b), the keratinocytes obtained in Example l(a) were inoculated in the same medium as in Example 1 and cultured at 37°C for 5 days at a humidity of 80-100% in 10% CO₂ atmosphere, followed by air exposure to induce differentiation of skin and hair-fbllicular keratinocytes. In detail, when reaching 100% confluency on the collagen layer, the keratinocytes submerged in the culture medium were exposed to air by layering highly absorbent cotton pads beneath the collagen layer to lower the medium level to the pads. Under this condition, culturing for three weeks led to differentiation of the keratinocytes, and the resulting skin model was fixed with 10% formalin and embedded in a paraffin block which was then sectioned and stained with H&E (Hematoxylin & Eosin)(FIGS.3A and 3B).

As is apparent from FIGS. 3A and 3B, the keratinocytes obtained in Example 1 were fully differentiated to form, from the innermost layer out, a basal layer, a spinous layer, a granular layer and a cornύied layer, regardless of the origin of their tissues.

EXAMPLE 3 : Comparison of Protein Expression Patterns of Keratinocytes over Culture Periods

0.2x10⁶ keratinocytes were inoculated onto a 313 feeder and cultured for 8, 9 and 10 days, and some of the cells cultured for 10 days were freeze-dried. The resultant keratinocytes were separated into single cells by treatment of trypsin:EDTA and the resulting cell suspension was treated with a lysis buffer to extract proteins which were then dissolved in a sample buffer containing SDS (Sodium Dodecyl Sulphate) and heated. The denatured proteins were separated through 10% SDS-PAGE and visuali2MvΛthCoommassieblue(FIG.4).

As seen in FIG. 4, the expression pattern and amount of protein in keratinocytes were constant over time period of culture and exhibited almost no difference before and after lyophilization (lanes 3 and 4). Accordingly, lyophilization is found to have no influence on the protein expression and secretion of the keratinocytes. EXAMPLE 4: Comparison of Protein Expression Patterns of Fibroblasts over Culture Periods

When grown at 50%, 70%, 90% and 100% confluency, skin fibroblasts were separated and then analyzed for protein expression pattern on SDS-PAGE as in Example 3 (FIG. 5, lanes 2 to 5). Concurrently, when the fibroblasts reached 50%, 70%, 90%, and 100% confluency, a portion of their media was used to analyze the expression pattern of protein in the cells on SDS-PAGE (FIG. 5, lanes 6 to 9).

As shown in FIG. 5, the patterns of the proteins extracted from the fibroblasts were identical irrespective of the length of culture period, but dϋferent from those of the proteins secreted into the culture media Ih addition, the proteins obtained both from the cells and from the culture media exhibited patterns different from those of the proteins obtained from the keratinocytes. As was understood from the similar result from the analysis of 10% serum alone (FlG. 5, lane 1), it was difficult to read a change in the protein patterns of the culture media However, it was found that as the confluency increased, two bands of the size between 120 and 200 KD became thickened Based on the size, these bands were Hiought to represent collagen. The analysis of the protein pattern obtained in the serum-free culture media indicated that all proteins including collagen increased in quantity with an increase in confluency.

EXAMPLE 5: Comparison of Amounts of Cytokines Secreted from Keratinocytes and Fibroblasts

Cytokines secreted from keratinocytes and fibroblasts before and after the lyophilization thereof were analyzed using ELISA. Ih detail, with the help of Ihe Quantikine Immunoassay Kit, manufactured by R&D System, IL-lα, bFGF, TGF-β and PDGF, secreted from the cells, were quantitatively analyzed (FIG.6). As seen in FIG. 6, the cytokine amount from the live cells before the lyophilization was found to be similar to that from the dead cells after the lyopbilization, indicating that lyophilization had no influence on the secretion of the cytokines.

EXAMPLE 6: Comparison of Colony Forming Efficiency of Keratinocytes

About 100 cells of primarily cultured keratinocytes were seeded on a 3T3 feeder and cultured for 12 days. The lyophilized keratinocytes or fibroblasts were suspended in keratinocyte culture medium at a concentration of 0.5^χl0⁶ cells/ml, which was used as a culture medium. Upon completion of culturing, the cells were fixed with 10% formalin and stained with Rhodamine blue. Colony forming efficiency (CFE) was expressed as a percentage of the number of colonies relative to the number of cells inoculated, as in Formula 1 , below. [Formula 1]

CFE % ^ ^Nθ- ^{θf Cθlθnies fθrmed} χ l00 No. of Cells inoculated

As seen in FIG. 7, when a culture medium containing no lyophilized cells was used (control), the cells showed a CFE of 35%, while culture media containing the lyophilized keratinocytes or fibroblasts allowed cells to exhibit CFEs of 46% and 44%, respectively. In particular, colony sizes were increased from 3.2 mm on a lyophilized cell-free medium to 8.3mm and 7.9mm on media containing lyophilized keratinocytes and fibroblasts, respectively. These data imply that keratinocyte- or fibroblast-containing culture media can supply cytokines necessary for the proliferation and migration of keratinocytes, resulting in increases in the number and size of colonies.

EXAMPLE 7: Comparison of Proliferation Rate of Fibroblasts

0.5^χl0⁵ fibroblasts were inoculated in each well of a 24-well plate containing DMEM supplemented with 10% fetal bovine serum therein. After culturing for 24 hours, the culture medium was replaced with DMEM supplemented with 5% FBS, followed by suspending lyophilized keratinocytes or fibroblasts at a density of 0.5 ^χ 10⁵/ml in the culture medium. After culturing for 24 hours, fibroblasts were assayed for proliferation rate using MTT (telrazolium salt 3-[4,5- dimethylthiazoI-2-yl]-2,5-diphenyltetrazolium bromide]). In detail, an MTT solution (5mg/ml in PBS) was added to each well of the well plate and reaction was allowed for 4 hours at 37⁰C in a humidified atmosphere. After careful removal of the culture medium, a lysis buffer containing 10% SDS and 45% dimethyl foramide (pH4.7) was added to the wells and reacted with the cells for 12 to 24 hours. The mitochondria's ability to reduce yellow water-soluble MTT tetrazolium to violet water- insoluble MTT Foiinazon(3-(4,5-dimethyltWa2θl-2-yl)-2,5-diphenyl-tetrazOHιim bromide) through dehydrogenaπ^'on, that is, the proliferation rate of cells, was determined by measuring absorbance at 590nm(FIG. 8).

As seen in FIG. 8, culture media containing keratinocytes or fibroblasts increased the proliferation rate of fibroblasts by 21 to 30%, compared to a culture medium containing neither keratinocytes nor fibroblasts. Accordingly, lyophilized kerafinocytes or fibroblasts can promote the proliferation of fibroblasts as well as keratinocytes, as in Example 6.

EXAMPLE 8: Comparison of Fibroblasts' Ability to Produce Collagen

To determine whether keratinocytes or fibroblasts contained in the cosmetic composition can promote skin regeneration or not, collagen, one of the extracellular matrices that fibroblasts express, was quantitatively assayed. 500 μl of a conditioned medium collected during cultivation under the same condition as in Example 7 was mixed with 1 ml of Sirius Red, followed by the measurement of absorbance at 540 nm (FIG.9). Culture media containing keratinocytes and fibroblasts, as shown in FIG. 9, were found to allow 35.4% and 25.6% more collagen, respectively, to be produced relative to a culture medium containing neither keratinocytes nor fibroblasts (control).

EXAMPLE 9: Stability of Composition Comprising Keratinocytes and Fibroblasts

To examine whether the keratinocytes in a composition can secrete proteins steadily over time so as to maintain the efficacy of skin regeneration, DL- lα, one of the factors secreted from keratinocytes, was quantitatively analyzed. This may provide a criterion to determine the shelf life during which the composition containing the cells keeps its efficacy (FIG. 10). Even 8 weeks after its preparation, as seen in FIG. 10, the level of IL-I α in the composition containing keratinocytes was maintained at 70% or higher relative to that just after preparation.

EXAMPLE 9: Preparation of Cosmetic Composition Comprising Keratinocytes and Fibroblasts

The cosmetic composition comprising lyophilized keratinocytes and fibroblasts according to the present invention was formulated in the form of an essence in order to examine the activity and safety of cytokines secreted from the cells contained in the formulation when applied to human skin.

TABLE l

Cosmetic Compositions Comprising keratinocytes/ Fibroblasts

In a vessel were placed the components of Group A which were then mixed using a mixer to give an aqueous phase. The components of Group B were slowly added to the aqueous phase and sufficiently stirred to homogeneity. The components of Group C were homogenized by heating (5O⁰C) in a separate vessel and slowly fed into the homogenized mixture of Groups A and B with stirring. As a result, micelles were formed. Thereafter, the component of Group D was added to make the composition neutral and viscous. Ih the case of Composition B, the component of Group E was added to prepare an essence-type cosmetic in a viscous state.

EXAMPLE 10: Test of Cosmetic Composition Comprising Keratinocytes and Fibroblasts for Skin Regeneration The composition (A) or (B) prepared in Example 9 was applied to volunteers suffering from atopic dermatitis in order to assay it for skin regeneration ability through the quantitative analysis of water content in the cornified layer and the epidermis.

30 volunteers who had mild cases of atopic dermatitis (that is, over whose entire skin atopy was, not severely but slightly, spread) were selected (hereinafter referred to as 'examinees'). Prior to the application of the cosmetic composition of the present invention to the examinees, their skin was photographed, measured for skin hydration and transepidermal water loss (TEWL), and their apparent symptoms were recorded On the basis of these measurements, two sites (hereinafter referred to as 'test site') that showed similar skin hydration and TEWL values within tolerance among the examinees were selected. Individual examinees themselves applied the composition (A) or (B) onto their test sites twice per day.

Skin hydration and TEWL were measured once per week for six weeks (before application and after application for 1, 2, 4, and 6 weeks: a total of 5 times). Before measurement, the test sites were cleaned without irritation and acclimatized for 30 min or more in temperature and humidity- controlled room (20⁰C, humidity 50%).

(a) Measurement of Skin hydration

Using a corneometer (C&K), test sites were measured for skin hydration (corneometer unit) and the results are given in Table 2, and FIG. 11.

TABLE 2

Skin Hydrations Measured

As is apparent from the data of Table 2, skin hydration increased with an increase in the length of application of the compositions. It was also found that composition (B) could maintain skin hydration better than composition (A).

A statistical analysis shows a significant difference between skin hydrations of compositions (A) and (B) (p=0.011)₅ but no significant difference among skin hydrations over the length of application of each composition (p=0.09664). The skin hydration, although statistically insignificant, increased with the number of applications of the cosmetic composition, and was measured to be approximately 23.5% higher after 6 weeks of application than that at the time of application.

(b) Measurement of TEWL Using a tewameter, the test sites were measured for TEWL in gtaVh unit and Hie results are given in Table 3 and FIG. 12. TABLE 3

Transepidermal Water Loss Measured

As seen in Table 3, TEWL was higher in composition (A) than composition (B). Upon the application of composition (B), the TEWL increased from 8.14 in week zero to 10.17 in week one, but started to decrease more rapidly from week two. Composition (A) increased skin hydration from 21.16 in week zero to 25.09 in week one, and decreased skin hydration thereafter.

The results of statistical analysis demonstrate that there is a significant difference between TEWL of compositions (A) and (B) (p=0.0055), but no significant difference among TEWLs over the length of application of each composition (p=0.4373). The TEWL, although statistically insignificant, decreased with the number of applications of the cosmetic composition, and was measured to be approximately 13.4% lower after 6 weeks of application than in week zero.

INDUSTRIAL APPLICABILITY

Taken together, the data obtained in the Examples demonstrate that the keratinocytes and/or fibroblasts, even if combined with a cosmetically acceptable carrier, can induce rapid skin rejuvenation or regeneration.

Claims

1. A cosmetic composition, comprising at least one of lyophilized keiaώiocytes and lyophilized fibroblasts.

2. The cosmetic composition as defined in claim 1, wherein the keratinocytes or the fibroblasts secrete cytokines selected from a group consisting of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), keratinocyte growth factor (KGF), transforming growth factor alpha (TGFα), transforming growth lactor beta-1 (TGFβ), granulocyte colony stimulating factor (GCSF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF), interleukins, and combinations thereof.

3. The cosmetic composition as defined in claim 1, wherein the keratinocytes are contained in an amount of 0.01 to 2.0 % by weight based on the total weight of the compositioa

4. The cosmetic composition as defined in claim 1 , wherein the fibroblasts are contained in an amount of 0.01 to 5.0 % by weight based on the total weight of the compositioa

5. The cosmetic composition as defined in claim 1, wherein the keratinocytes are derived from differentiated skin, sldn appendages or embryonic stem cells.

6. The cosmetic composition as defined in 5, wherein the skin is selected from a group consisting of foreskin, head skin, skin from the armpit, the hips, the breasts, the pubis, and the scrotum, and combinations thereof.

7. The cosmetic composition as defined in claim 5, wherein the skin appendage is selected from a group consisting of hair follicles, sweat glands, sebaceous glands, and capillary vessels.

8. The cosmetic composition as defined in claim 1, wherein the fibroblasts are derived fiυm differentiated skin or embryonic stem cells.

9. The cosmetic composition as defined in claim 1, wherein the composition is in a cosmetic water, lotion, cream, or gel form.

10. A method of preparing a cosmetic composition of one of claims 1 to 9, comprising:

(1) culturingkeratinocytes or fibroblasts;

(2) freeze-drying the cultured keratinocytes or fibroblasts; and

(3) incorporating the freeze-dried keratinocytes or fibroblasts into a cosmetically acceptable carrier.