KR101986092B1 - Method for Isolation of high purity papillary dermal fibroblasts from fibroblasts - Google Patents

Abstract

The present invention relates to a method for separating papillary dermis fibroblasts and a method for obtaining a high purity papillary dermis fibroblast population. The method for separating papillary dermis fibroblasts according to the present invention and the method for collecting high purity papillary dermis fibroblasts can highly isolate the papillary dermis fibroblast part having excellent skin regeneration effect from the fibroblasts. In addition, the composition comprising the papillary dermis fibroblast and the population thereof separated by the above method is superior to the conventional fibroblast treatment agent and has excellent skin regeneration effect, so that it can be used not only as a medicine but also as a quasi-drug or a cosmetic .

Description

TECHNICAL FIELD The present invention relates to a high purity papillary dermal fibroblast from fibroblasts,

The present invention relates to a method for separating papillary dermis fibroblasts and a method for obtaining a high purity papillary dermis fibroblast population.

The pharmaceutical market is developing from chemical synthetic drugs to biopharmaceuticals. Biopharmaceuticals are drugs made from raw materials or materials such as proteins, genes and cells derived from human or other organisms. They can be divided into antibody drugs, cell therapy drugs, and biosimilars.

 Unlike protein drugs, cell therapy is a new technology starting from the principle of treating the root cause of a disease. Recently, studies on scar treatment using fibroblasts, skin burn treatment using dermal keratinocyte, and arthritis treating agent using stem cells have been conducted, and product development using such agents has been actively carried out. The existing autologous fibroblast treatment agent is being used as a method for firstly separating fibroblasts from the dermal tissue of the patient's ear and then proliferating and culturing it to apply to the patient's skin. It is used for skin depression, wrinkles and intractable dermatitis . ≪ / RTI > However, it has been reported that the skin regenerating ability of the fibroblast treatment agent for skin depression is inferior in activity to the conventional therapeutic method and beauty filler. In order to overcome this difference in function, an additive such as hyaluronic acid or a polymer or a low molecular weight compound scaffold is added in addition to the autologous fibroblast, but the functional improvement is not achieved.

On the other hand, according to recent studies, fibroblasts are classified into papillary dermis fibroblasts and reticular dermis fibroblasts according to their locations, and it is confirmed that there are differences in function and activity. Nevertheless, most of the autologous fibroblast treatments currently on sale and research are using fibroblasts in which various cells are mixed, and its efficacy or efficacy is insufficient.

The present inventors have judged that the lack of efficacy or effectiveness of the fibroblast treatment agent is due to the heterogeneity of fibroblasts as raw materials. Accordingly, the present inventors have made intensive efforts to separate papillary dermis fibroblasts having excellent skin regeneration effect from fibroblasts with high purity, and as a result, it has been confirmed that the papillary dermis fibroblasts can be easily and rapidly separated using the transwell culture method Thus completing the present invention.

It is an object of the present invention to provide a separation method for culturing fibroblasts mixed in a transwell and obtaining cells present at the upper part of the upper chamber to separate the papillary dermis fibroblasts.

It is another object of the present invention to provide a method for culturing mixed fibroblasts in transwells and obtaining cells present at the top of the upper chamber to obtain a population of highly purified papillary dermal fibroblasts.

Another object of the present invention is to provide a pharmaceutical composition for skin regeneration, a quasi-drug composition, and a cosmetic composition comprising the group of mammary gland fibroblasts obtained by the above method.

In order to achieve the above object, the present invention provides a method for producing fibroblasts, comprising: (1) obtaining fibroblasts from skin tissue; (2) inoculating the transwell with the obtained cells; (3) culturing the inoculated cells; And (4) obtaining cells present on top of the transwell upper chamber; The present invention also provides a method for separating papillary dermal fibroblasts.

The present invention also relates to a method for producing fibroblasts, comprising: (1) obtaining fibroblasts from skin tissue; (2) inoculating the fibroblasts into transwells; (3) culturing the inoculated cells; And (4) separating cells present on top of the transwell upper chamber; Papillary dermal fibroblast < / RTI > population.

The present invention also provides a pharmaceutical composition for skin regeneration comprising the group of mammary gland fibroblasts obtained by the above method.

In addition, the present invention provides a quasi-drug composition for skin regeneration comprising the group of mammary gland fibroblasts obtained by the above method.

The present invention also provides a cosmetic composition for skin regeneration comprising the group of mammary gland fibroblasts obtained by the above method.

The method for separating papillary dermis fibroblasts according to the present invention and the method for collecting high purity papillary dermis fibroblasts can highly isolate the papillary dermis fibroblast part having excellent skin regeneration effect from the fibroblasts. In addition, the composition comprising the papillary dermis fibroblast and the population thereof separated by the above method is superior to the conventional fibroblast treatment agent and has excellent skin regeneration effect, so that it can be used not only as a medicine but also as a quasi-drug or a cosmetic .

1 shows the results of comparing the expression amounts of papillary dermal fibroblast marker NTN1 and reticulated dermal fibroblast marker MGP according to the number of cells inoculated with fibroblasts and the top and bottom positions of the upper chamber of the transwell .
2 is a schematic diagram of a method for separating papillary dermal fibroblasts according to the present invention.
FIG. 3 is a diagram showing the results of confirming the cell proliferation capacity according to the top and bottom positions of the transwell upper chamber during 0 to 9 days of culturing. FIG.
FIG. 4 is a graph showing the results of comparing the expression levels of nasopharyngeal fibroblast marker NTN1 and reticuloendothelial fibroblast marker MGP according to the number of transwell isolation times.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for separating papillary dermis fibroblasts, which comprises a transwell culturing step.

Specifically, the method for separating the papillary dermal fibroblasts of the present invention comprises the steps of: (1) obtaining fibroblasts from skin tissue; (2) inoculating the transwell with the obtained cells; (3) culturing the inoculated cells; And (4) obtaining cells present on top of the transwell upper chamber; And a method for separating papillary dermal fibroblasts.

According to the separation method of the present invention, only the papillary dermis fibroblast can be selectively isolated with high purity, and only the obtained papillary dermis fibroblast can be cultured in a large amount, and only the papillary dermis fibroblast is highly purified, Can be further increased.

(1) of the present invention is a step of obtaining fibroblasts from skin tissue. The step of obtaining such a fibroblast can be carried out by a conventional method known in the art. For example, after washing the skin tissue, the dermal layer of the skin is separated, the dermal layer is fixed on the culture plate, and the cells are dissociated to obtain a fibroblast population. At this time, the skin tissue can be obtained without restriction from various parts of the body, and the individual for obtaining the skin tissue is preferably a mammal including human.

As used herein, the term " fibroblast " is a cell that forms an important component of fibrous connective tissue, and is also referred to as a fibroblast. Observed with tissue section, it has a flat and elongated appearance, often irregular protrusion, cytoplasm contains mitochondria, Golgi body, central body, small lipid body, and other special differentiation is not shown. The nucleus is weakly dyed, oval, and contains phosphorus. In many cases, it is closely related to the collagen fibers, and it is considered that there is a relationship with the formation, so that the above-mentioned name is attached.

The fibroblasts obtained in step (1) of the present invention may be in a mixed state including both papillary dermal fibroblast and reticular dermal fibroblast.

The fibroblasts are divided into two groups according to the location of the upper and lower parts of the dermis tissue: Papillary dermal fibroblast and Reticular dermal fibroblast. Here, the papillary dermis is a thin area beneath the epidermis, and the thick part from the papillary dermis to the subcutaneous fat layer surface can be classified as reticular dermis. The papillary dermis and the reticular dermis are different in size and shape, and have different functional characteristics. For example, the collagen of the papillary dermis is present as a net composed of a thin fiber, while the collagen of the reticular dermis forms a thick bundle, As shown in FIG.

Step (2) of the present invention is a step of inoculating the transwell with the obtained cells.

As used herein, the term "transwell" or "transwell plate" is widely used in the art, and a commercially available product can be used. Information on the transwell plate and how to use it can be obtained by a person skilled in the art It is within the skill level. The transwell plate is composed of a top chamber having a bottom portion made of a microporous membrane and a bottom chamber containing a medium, the top chamber being located inside the bottom chamber, and the two chambers sharing a culture medium . During transwell incubation, additional components may be included and may include additional substrates to achieve the objectives of the present invention, such as keratinocyte secretion factor or epidermal growth factor growth factor, EGF), and the like.

Here, the microporous membrane is a support through which a cellular material such as a cell secretory material, a culture medium, and a stem cell can permeate, and the size of the hole of the microporous membrane can be appropriately adopted by those skilled in the art. The pore size of the microporous membrane is preferably such that the mixed dermal fibroblasts located in the upper chamber do not permeate all the way into the lower chamber. For example, the pore size of the microporous membrane is 1 to 10 mu m, May be 5.0 to 10 mu m, more preferably 7.0 to 8.5 mu m.

As the microporous membrane constituting the bottom surface of the transwell upper chamber, a membrane made of a material commonly used in the art can be used. For example, polycarbonate, polyester, collagen-coated polytetrafluoroethylene, Can be used as a porous membrane.

When the fibroblasts obtained in the separation method (1) step of the present invention are inoculated into the transwell, the number of inoculated cells can be adjusted so as to be suitable for culturing, and the cells can be adjusted to a concentration of 1 x 10 ⁴ to 1 x 10 ⁶ cells, Preferably 1 x 10 ⁴ to 2 x 10 ⁵ cells, more preferably 0.2 x 10 ⁵ to 2 x 10 ⁵ cells.

The step (3) of the present invention is a step of culturing the inoculated cells, and the step (4) is a step of obtaining cells existing on the upper side of the transwell upper chamber.

The culture of the fibroblasts can be carried out according to conventional fibroblast culture conditions and is cultured for 1 to 5 days, preferably 2 to 5 days, more preferably for 3 days in a cell incubator under the condition of 37 ° C and 5% CO ₂ Lt; / RTI >

The skin fibroblasts mixed in the culture step are moved through the microporous membrane of the upper chamber, resulting in the accumulation of papillary dermis fibroblasts at the upper end of the upper chamber and the reticular dermis fibroblasts at the lower end of the upper chamber.

This migrated cell population can be identified by marker expression of Eudypidia fibroblasts or markers of reticulated dermal fibroblasts expressed at each location.

The marker may be a reporter gene. In one embodiment of the present invention, NTN1 (Netrin-1) is used as a marker of papillary dermal fibroblasts, and MGP (Matrix gla protein) is used as a marker of reticuloendothelial fibroblasts. But is not limited to.

The cell obtaining step of the present invention means collecting cells present on the top of the transwell upper chamber.

In the present invention, the transwell " upper chamber top " means the surface portion of the bottom of the upper chamber facing the cell culture space of the upper chamber. Transwell " upper chamber bottom " as used herein means the surface of the upper chamber bottom facing the space defined by the upper chamber and the lower chamber.

The papillary dermis fibroblasts obtained in the present invention are collected at the upper end of the transwell upper chamber through transwell culture and can be obtained by selecting only the cells located at the upper end of the transwell upper chamber for obtaining the desired cells , And only the obtained cells can be cultured to obtain a large amount of papillary dermal fibroblasts.

In addition, the step (2) to (4) of the present invention may be repeated 1 to 10 times, preferably 1 to 5 times, in order to select only the papillary dermis fibroblasts at a higher purity. In the case of repeatedly performing this step, the cells present at the top of the transwell upper chamber of step (4) are obtained, and then this is applied to step (2) to inoculate the transwell and the inoculated cells are cultured repeatedly can do.

According to the above method, only the desired papillary dermal fibroblast can be easily separated from the mixed dermal fibroblast.

Accordingly, the present invention provides a method for obtaining a population of high purity papillary dermal fibroblasts that can be achieved through the above separation method. More specifically, the present invention relates to a method for producing fibroblasts, comprising: (1) obtaining fibroblasts from skin tissue; (2) inoculating the fibroblasts into transwells; (3) culturing the inoculated cells; And (4) separating cells present on top of the transwell upper chamber; Papillary dermal fibroblast < / RTI > population.

According to the method of the present invention, reticulated dermal fibroblasts can effectively be obtained from a group of mixed dermal fibroblasts in which a highly pure papillary dermal fibroblast group is removed. Thus, in the cell population obtained according to the present invention, the proportion of papillary dermal fibroblasts is markedly increased and the proportion of reticulated dermal fibroblasts is markedly reduced compared to that before transwell culture.

In the present invention, high purity means that the presence of other cells such as reticulate dermal cells in the cell population is reduced and only the papillary dermal fibroblasts are present at a high rate. The criteria for reduction and increase are obtained from the skin tissue before transwell culture Lt; / RTI > fibroblast population.

In addition, the present invention provides a pharmaceutical composition for skin regeneration comprising the hair follicular dermal fibroblast obtained by the above method. The papillary dermis fibroblast of the present invention is an effective ingredient for promoting skin regeneration, and includes a culture solution thereof and the like.

As used herein, the term " skin regeneration " refers to an increase in the elasticity of the skin, an improvement in wrinkles, an inhibition of progress of aging, etc. When the composition of the present invention is applied to the skin, proliferation of fibroblasts is initiated by papillary dermal fibroblasts And reduces the death of existing cells, thereby increasing the elasticity of the skin and delaying and preventing the generation of wrinkles, thereby delaying aging.

The pharmaceutical compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The compositions of the present invention may be formulations that can be administered by a method such as direct application or dispensing to the skin, e.g., the hair or scalp, such as a formulation of a cream, lotion, ointment, aerosol, gel, have. Methods for formulation ingredients and formulations suitable for each formulation are well known in the art. Those skilled in the art can appropriately select and use various formulation ingredients used in the preparation of these external preparations when manufacturing these preparations.

Such components include, for example, ointments such as white petrolatum, yellow petrolatum, lanolin, bleached beeswax, cetanol, stearyl alcohol, stearic acid, hardened oil, gelled hydrocarbon, polyethylene glycol, liquid paraffin, Bases such as squalane; Solvents and dissolution aids such as oleic acid, myristic acid isopropyl, triisooctanoic acid glycerin, crotamiton, diethyl sebacate, diisopropyl adipate, hexyl laurate, fatty acid, fatty acid ester, aliphatic alcohol and vegetable oil; Antioxidants such as tocopherol derivatives, L-ascorbic acid, dibutylhydroxytoluene, and butylhydroxyanisole; Preservatives such as parahydroxybenzoic acid ester; Humectants such as glycerin, propylene glycol and sodium hyaluronate; Surfactants such as polyoxyethylene derivatives, glycerin fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, propylene glycol fatty acid esters, and lecithin; Carboxyvinyl polymer, xanthan gum, carboxymethylcellulose, carboxymethylcellulose sodium salt, hydroxypropylcellulose, hydroxypropylmethylcellulose and the like.

In the case of the aerosol preparation, the white petrolatum, the yellow petrolatum, the lanolin, the bleached wax, the cetanol, the stearyl alcohol, the stearic acid, the stearic acid, Base oils such as hydrogenated oils, gelled hydrocarbons, polyethylene glycols, liquid paraffin, squalane and the like; Oleic acid, myristic acid isopropyl, diisopropyl adipate, isobutyl sebacate, glycerin triisooctanoate, crotamiton, diethyl sebacate, hexyl laurate, fatty acid esters, aliphatic alcohols and vegetable oils Solvents and dissolution aids; Antioxidants such as tocopherol derivatives, L-ascorbic acid, dibutylhydroxytoluene, and butylhydroxyanisole; Preservatives such as parahydroxybenzoic acid ester; Humectants such as glycerin, propylene glycol and sodium hyaluronate; Surfactants such as polyoxyethylene derivatives, glycerin fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, propylene glycol fatty acid esters, and lecithin; Thickening agents such as carboxyvinyl polymer, xanthan gum, carboxymethyl cellulose, carboxymethyl cellulose sodium salts, hydroxypropyl cellulose, hydroxypropylmethyl cellulose and the like; In addition, various stabilizers, buffers, mating agents, suspending agents, emulsifiers, fragrances, preservatives, solubilizers, and other suitable additives may be added.

In addition, a stabilizer, a preservative, an absorption promoter, a pH adjuster, and other suitable additives may be added as necessary.

The composition of the present invention may be administered in a pharmaceutically effective amount.

The term " pharmaceutically effective amount " as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level will vary depending on the species and severity, age, sex, The type of drug, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition of the present invention may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art. The preferred dosage of the composition of the present invention will depend on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, and the appropriate total daily dose may be determined by treatment, in general, to 1.0X10 ³ 1.0X10 ¹⁰ Cell / positive ml, preferably from 1.0X10 ⁵ 1.0X10 ⁷ to be administered by dividing the amount of Cell / ml can to 1 per day circuit, and which the patient's condition and treatment As shown in FIG.

Meanwhile, the method of administering the pharmaceutical composition according to the present invention is not particularly limited, and preferably, it can be administered in the form of being applied to the skin region by the transdermal administration method. Preferably, And there is no particular limitation on the method of administration.

Also, the dosage level selected from the composition will depend on the activity of the active ingredient, the route of administration, the condition of the skin to be treated and the prior history. It is, however, within the knowledge of the art to gradually increase the dose until the desired effect is achieved, starting with a dose of the active ingredient at a level lower than that required to achieve the desired therapeutic effect, Sex, body shape, and body weight.

In addition, the present invention provides a quasi-drug composition for skin regeneration comprising papillary dermis fibroblasts isolated by the above method.

The term " quasi-drug product " in the present invention means a fiber, a rubber product or the like used for treating, alleviating, treating or preventing a disease of a human or an animal, a weak action on the human body, Or products similar to those which are not machinery, preparations used for sterilization, insecticides and similar uses for the prevention of infections, for the purpose of diagnosis, treatment, alleviation, treatment or prevention of diseases of human beings or animals Machinery, or apparatus, and that is not an apparatus, machine, or apparatus of an article used for the purpose of giving pharmacological effects to the structure or function of a person or animal, It also includes supplies.

When the composition of the present invention is contained in quasi-drugs for the purpose of regenerating skin, the composition may be used as it is or may be used together with other quasi-drugs, and may be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be appropriately determined depending on the purpose of use.

The external preparation for skin may be prepared and used in the form of, for example, an ointment, a lotion, a spray, a patch, a cream, a powder, a suspension or an ointment.

The present invention also provides a cosmetic composition for skin regeneration comprising papillary dermis fibroblasts isolated by the above method. The papillary dermis fibroblast of the present invention is an effective ingredient for promoting skin regeneration, and includes a culture solution thereof and the like.

The cosmetic composition may be formulated into a general cosmetic composition (for example, lotion, lotion, cream, gel, etc.) using a technique known in the art as an external preparation for skin. The type and concentration of the carrier that can be used depend on the desired formulation, but can be readily determined by one of ordinary skill in the art.

The 'cosmetically acceptable carrier' may be selected from the group consisting of purified water, oils, waxes, fatty acids, fatty alcohols, fatty acid esters, surfactants, humectants, thickening agents, antioxidants, viscosity stabilizers, chelating agents, buffers, preservatives, lower alcohols, and the like. The cosmetic composition according to the present invention may contain a whitening agent, a moisturizing agent, an anti-inflammatory agent, an antibacterial agent, an antifungal agent, a vitamin, an ultraviolet screening agent, an antibiotic, an anti- . 0.01 to 20% by weight, based on the total weight of the composition, is suitable.

Specific examples of the wax include waxes such as beeswax, wax, carnauba, candelilla, montan, ceresin, liquid paraffin, lanolin, Can be used. Examples of the fatty acid include stearic acid, linoleic acid, linolenic acid and oleic acid. Fatty acid alcohols include cetyl alcohol, octyldodecanol, oleyl alcohol, panthenol, lanolin alcohol, stearyl alcohol and hexadecanol, As the ester, isopropyl myristate, isopropyl palmitate, and butyl stearate may be used, but the present invention is not limited thereto.

Examples of the surfactant include anionic surfactants such as sodium stearate, sodium cetyl sulfate, polyoxyethylene lauryl ether 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.

As the moisture absorbent, glycerin, 1,3-butylene glycol and propylene glycol may be used, and as the lower alcohol, ethanol and isopropanol may be used. Examples of thickeners include sodium alginate, sodium caseinate, gelatin agar, xanthan gum, starch, cellulose ethers (e.g., hydroxyethylcellulose, methylcellulose, carboxymethylcellulose, hydroxypropylmethylcellulose), polyvinylpyrrolidone, Polyvinyl alcohol, polyethylene glycol, sodium carboxymethylcellulose, and the like, but are not limited thereto.

As the antioxidant, butylated hydroxytoluene, butylated hydroxyanisole, propyl gallate, citric acid, and ethoxyquin are usable. As the chelating agent, disodium edetate, ethanhydroxy diphosphate And buffering agents such as citric acid, sodium citrate, boric acid, borax, and disodium hydrogen phosphate are available, and preservatives include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, dihydroacetic acid , Salicylic acid, benzoic acid, and the like.

In addition, the composition for skin regeneration of the present invention can be used for pet animals in different dosage forms. For example, a neutral detergent which can be manufactured in various forms such as a solution shampoo for pet and a rinse for pet such as solution, sol gel, emulsion, oil, wax, and aerosol, and is less irritating to pet skin and excellent in moisture And the like.

The present invention will be described in more detail by way of examples. It is to be understood, however, that the invention is not to be limited to the details given herein, and that, unless otherwise defined in the technical and scientific terms used, Have a general meaning to those of ordinary skill in the art.

Example  One. Obtaining fibroblasts

After washing the human foreskin or lid tissue with DPBS (Dulbecco's Phosphate-Buffered Saline), the dermis of the skin was separated by removing the fat inside the skin tissue with scissors and a razor blade. The isolated tissue was chopped and fixed for 5 to 15 minutes to attach the dermis to the bottom of the 100 mm culture plate. Fixed tissues were fed with DMEM (Dulbecco Modified Eagle Medium) containing 10% fetal bovine serum and 1% penicillin antibiotics in sufficient tissue lock (approximately 10 ml). Thereafter, after confirming that the cells disassociated from the tissue and adhered to and proliferated on the plate for a period of 2 to 4 weeks, when a sufficient amount of cells dissociated, the tissue was removed from the plate, and 0.25% trypsin EDTA (ethylenediaminetetra acetic acid) To obtain mixed fibroblasts.

Example  2. Transwell  Used Papilla dermis  Isolation of fibroblasts

2.1 Transwell  Isolation of cells used

In order to separate the papillary dermis fibroblast and the reticulated dermis fibroblast from the mixed fibroblast group obtained in Example 1, a group of fibroblasts mixed in the transwell was placed and the effect of the separation was observed. Specifically, the mixed fibroblast populations were inoculated into transwells (falcon, 353093, 8.0 μm pore size (1 × 10 ⁵ pores / cm ² )) having 6 well plates and cultured. As the culture medium, medium supplemented with 10% fetal bovine serum 1% penicillin streptomycin in WELGENE's DMEM high glucose was used. As a control group, 1 × 10 ⁵ cells were inoculated in a general 6-well culture range, and 0.2, 0.4, 1, 2 × 10 ⁵ cells were inoculated into the upper chamber of the transwell as test groups. After inoculation, the cells were cultured in a cell incubator at 37 ° C and 5% CO ₂ for 3 days. The top and bottom layers of the upper chamber of the control and transwell test groups were then obtained using 0.25% trypsin EDTA (ethylenediaminetetraacetic acid).

2.2 Transwell  Confirmation of cell separation effect

In order to confirm whether the obtained cells of each site were effectively separated from the papillary dermis fibroblasts and reticulated dermis fibroblasts, marker expression was analyzed in cells obtained from each site. RNA was first isolated from cells obtained at each site using the High pure RNA isolation kit (Roche, 11828665001). CDNA was then synthesized from the RNAs isolated using the Transcriptor first strand cDNA synthesis kit (Roche, 4897030001). RT-PCR was performed on the isolated and synthesized cDNA, and the expression of NTN1 (Netrin-1), a marker of papillary dermis fibroblast, and MGP (Matrix gla protein), a marker of reticulated dermal fibroblast were quantified as EI24 gene And analyzed by relative quantification. The primer sequences used for RT-PCR are as follows, and the results of comparing the marker expression are shown in Fig.

As shown in FIG. 1, an increase in NTN1, a marker of papillary dermis fibroblasts, and a decrease in MGP, a marker of reticular dermis fibroblasts, were observed in cells isolated from the upper part of the transwell upper chamber. In contrast, cells isolated from the lower chamber of the transwell showed decreased NTN1 and increased MGP. These results indicate that the mixed fibroblasts can be separated into papillary dermal fibroblasts and reticulated dermal fibroblasts by transwell culturing. Fig. 2 is a schematic diagram of a method for separating dermal fibroblasts using the transwell according to the present invention.

Example  3. Isolated Papilla dermis  Estimation of proliferation of fibroblasts

The proliferative potential difference of the cells isolated by the method of Example 2 was evaluated. Cells at the upper and lower chambers of the upper chamber were obtained, respectively, and mixed fibroblast (none), upper cell (top), and lower cell (bottom), which were not cultured in the transwells, were subcultured. As the culture medium, medium supplemented with 10% fetal bovine serum and 1% penicillin streptomycin in DMEM high glucose of WELGENE was used. 5 × 10 ³ cells were inoculated in a 60 mm dish and cultured at 37 ° C. in a CO ₂ incubator at a constant temperature and humidity of 5%. The culture medium was replaced at intervals of 2 to 3 days, and suspended with trypsin EDTA 0.25% at 3, 6, and 9 days from the day of cell inoculation. The obtained cells were counted to measure the number of cells, and the proliferation ability of the cells was evaluated. The results are shown in FIG.

 As shown in Fig. 3, the proliferation rate of the cells isolated from the upper chamber top was superior to the cells in the control without transwell, and superior to the cells isolated from the bottom. On the other hand, the proliferation rate of the cells isolated from the lower end was found to be smaller than that of the control cells. These results show that the cells are superior to the dermal dermal fibroblasts in the mixed state of the separated papillary dermal fibroblasts at the top of the upper chamber separated by the culture method using the transwell and the dermal dermal fibroblasts at the lower stage. That is, by using the transwell culture method, it is possible to effectively separate and select only the papillary dermis fibroblasts having excellent proliferation power from the mixed dermis fibroblasts, and when using the separated papillary dermis fibroblasts, compared to using the mixed dermis fibroblasts Thereby further promoting skin regeneration.

Example  4. Transwell  Repeated cell separation

Since it was confirmed that the papillary dermis fibroblast and the reticulated dermis fibroblast can be effectively separated by the transwell culture, optimal separation conditions were confirmed by confirming whether or not the separation yield was changed according to the number of times of transwell isolation. For this purpose, the number of separation using transwell was changed to 0, 1, 2, and skin fibroblasts were cultured. The expression levels of markers NTN1 and MGP in the cell population corresponding to the number of separation were confirmed and compared. RT-PCR for confirmation of the expression level was carried out according to the method of Example 2, and the results are shown in Fig.

As shown in Fig. 4, the separation yield increased as the number of times of transwell isolation was increased. These results support the possibility that the dermal fibroblasts can be separated into papillary dermal fibroblasts and reticulated dermal fibroblasts by transwell culture.

Claims (10)

(1) obtaining fibroblasts from skin tissue;
(2) inoculating the obtained cells into a transwell having a pore size of 5.0 to 10.0 占 퐉;
(3) culturing the inoculated cells in DMEM high glucose medium containing fetal bovine serum; And
(4) obtaining cells present on top of the transwell upper chamber; Wherein the papillary dermal fibroblasts are separated from the papillary dermal fibroblasts.
The method according to claim 1,
Wherein the fibroblasts in step (1) comprise papillary dermis fibroblasts and reticulated dermis fibroblasts.
delete The method according to claim 1,
Wherein the cells of step (2) are inoculated at 1 × 10 ⁴ to 2 × 10 ⁶ cells.
The method according to claim 1,
The method for separating papillary dermal fibroblasts according to (3), wherein the culturing is performed for 1 to 5 days.
The method for separating papillary dermal fibroblasts according to claim 1, wherein the steps (2) to (4) are repeated one to five times.
(1) obtaining fibroblasts from skin tissue;
(2) inoculating the fibroblasts into a transwell having a pore size of 5.0 to 10.0 占 퐉;
(3) culturing the inoculated cells in DMEM high glucose medium containing fetal bovine serum; And
(4) separating cells present at the top of the transwell upper chamber; Lt; RTI ID = 0.0 > papillary < / RTI > dermal fibroblast population.
(1) obtaining fibroblasts from skin tissue;
(2) inoculating the fibroblasts into a transwell having a pore size of 5.0 to 10.0 占 퐉;
(3) culturing the inoculated cells in DMEM high glucose medium containing fetal bovine serum; And
(4) separating teat dermis fibroblasts existing on top of the transwell upper chamber; Wherein the skin elasticity is increased or wrinkle is improved.
(1) obtaining fibroblasts from skin tissue;
(2) inoculating the fibroblasts into a transwell having a pore size of 5.0 to 10.0 占 퐉;
(3) culturing the inoculated cells in DMEM high glucose medium containing fetal bovine serum; And
(4) separating teat dermis fibroblasts existing on top of the transwell upper chamber; Wherein the composition is a mixture of at least two kinds of quasi-drugs.
(1) obtaining fibroblasts from skin tissue;
(2) inoculating the fibroblasts into a transwell having a pore size of 5.0 to 10.0 占 퐉;
(3) culturing the inoculated cells in DMEM high glucose medium containing fetal bovine serum; And
(4) separating teat dermis fibroblasts existing on top of the transwell upper chamber; Of the cosmetic composition for skin regeneration.

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