CN101351217A - Pharmaceutical compositions for cell therapy of pigmentation disorders - Google Patents
Pharmaceutical compositions for cell therapy of pigmentation disorders Download PDFInfo
- Publication number
- CN101351217A CN101351217A CNA2006800503254A CN200680050325A CN101351217A CN 101351217 A CN101351217 A CN 101351217A CN A2006800503254 A CNA2006800503254 A CN A2006800503254A CN 200680050325 A CN200680050325 A CN 200680050325A CN 101351217 A CN101351217 A CN 101351217A
- Authority
- CN
- China
- Prior art keywords
- cell
- melanocyte
- horn cell
- fibroblast
- skin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/56—Materials from animals other than mammals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
- C12N5/0698—Skin equivalents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/09—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells
- C12N2502/091—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells melanocytes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/09—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells
- C12N2502/094—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells keratinocytes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
- C12N2502/1323—Adult fibroblasts
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
- C12N2533/52—Fibronectin; Laminin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
- C12N2533/54—Collagen; Gelatin
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Dermatology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Immunology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Developmental Biology & Embryology (AREA)
- General Chemical & Material Sciences (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Cosmetics (AREA)
Abstract
The present invention relates to a pharmaceutical composition for cell therapy of pigmentation disorders, which comprises as an active ingredient (a) melanocytes and keratinocytes, or (b) melanocytes and fibroblasts. The cell therapy composition of this invention allows for the first time to treat pigmentation disorders through cell therapy regimen. The present composition comprises as active ingredients a mixed cell preparation of melanocytes and keratinocytes or a mixed cell preparation of melanocytes and fibroblasts such that it induces very effectively pigmentation to treat diseases associated with hypopig mentation such as vitiligo.
Description
Technical field
The present invention relates to be used for the treatment of the drug cell therapy compositions and the painted skin reconstruction model of pigmentation disorder.
Background technology
Be used for comprising chemotherapy and surgical treatment as the therapy of the pigmentation disorder of white macula.Chemotherapy is usually by to dermal administration or local coating steroid, perhaps carries out to patient's photosensitizer administration and irradiation ultraviolet radiation.Yet this therapy may be induced lower pigmentation again and side effect.Surgical treatment also will normally carry out to the epidermic grafting of cut-out from the body surface skin grafting dermepenthesis by the epidermis of excision performance white macula symptom.
Korean Patent No.293736 has disclosed the Cortex Meliae that comprises carbohydrate-binding protein matter (Meliae Cortex) extract that is used for the treatment of white macula.Korean patent application No.2003-0056823 has disclosed the photosensitizer administration that is used for the treatment of white macula and the photodynamic therapy of local irradiation chemical light.In addition, korean patent application No.2003-7017182 has proposed to prove as has alleviated the system and method that living tissue light is regulated that is used for to skin beneficiating of white macula.Korean patent application No.2003-0049725 has reported the ultraviolet with 310nm wavelength of solid-state laser radiating system with the dermatosis that produces effective treatment such as psoriasis and white macula.
The application has quoted a plurality of patents and publication from start to finish, and citation is provided in bracket.These patents and publication are all introduced the application's technical merit more fully to describe the present invention and the present invention relates to as a reference at this.
Summary of the invention
The inventor has carried out further investigation to research and develop a kind of pigmentation disorder or disease of being used for the treatment of, particularly, the therapy of the cell therapy of the hypopigmentation symptom relevant with melanocyte forfeiture or melanocyte generation reduction found that melanocyte adds that horn cell or fibroblastic cell mixing preparation have greatly strengthened the pigmentation in the skin.
Therefore, an object of the present invention is to provide a kind of pharmaceutical composition that is used for the cell therapy of pigmentation disorder.
Another object of the present invention provides a kind of painted skin reconstruction model.
With claims and accompanying drawing, other purposes of the present invention and advantage will become apparent from the following detailed description.
An aspect of of the present present invention provides the pharmaceutical composition of the cell therapy that is used for pigmentation disorder, described pharmaceutical composition comprise (a) melanocyte and horn cell or (b) melanocyte and fibroblast as active component.
Another aspect of the present invention provides the method that is used for the treatment of pigmentation disorder, described method comprise use to the experimenter (i) melanocyte that comprises (a) pharmacy effective dose and horn cell or (ii) melanocyte and fibroblast as active component; (b) pharmaceutical composition of pharmaceutically suitable carrier.
Another aspect of the present invention provides and will comprise (i) melanocyte and horn cell or (ii) melanocyte and fibroblastic cell mixture are used to prepare the purposes of the medicine that is used for the treatment of pigmentation disorder.
The inventor has carried out further investigation to research and develop a kind of pigmentation disorder or disease of being used for the treatment of, particularly, the therapy of the cell therapy of the hypopigmentation symptom relevant with melanocyte forfeiture or melanocyte generation reduction found that melanocyte adds that horn cell or fibroblastic cell mixing preparation have greatly strengthened the pigmentation in the skin.
Marked feature of the present invention is to use simultaneously melanocyte to add that horn cell or fibroblast are used for the cell therapy of pigmentation disorder.The horn cell of being transplanted in the skin adheres to and breeds necessary somatomedin and promote the regeneration that melanocyte is attached to basement membrane and promotes epidermis as the intercellular substance of the component of basement membrane by providing melanocytic, causes producing more melanocyte and has strengthened pigmentation.The fibroblast of being transplanted in the skin with melanocyte promotes the formation of hemi desmosome (join domain in melanocyte or the horn cell basement membrane) and promotes basement membrane by providing as the intercellular substance of basement membrane composition, thereby strengthened painted again epidermis and the combination between the corium and improved the mechanical strength of skin.
According to embodiment preferred, melanocyte, horn cell and/or fibroblast are isolated from skin, or isolate and In vitro culture from skin.
According to embodiment preferred, the cell mixing preparation that is used as active component comprises melanocyte and horn cell.More effectively induce pigmentation by the cell mixing preparation that melanocyte and horn cell are formed than the cell mixing preparation of forming by melanocyte and fibroblast.More preferably, the active component of described compositions comprises three kinds of cell types, i.e. melanocyte, horn cell and fibroblast.
According to embodiment preferred, be leukoderma, pityriasis alba, nevus depigmentation, postinflammatory depigmentation or white macula, the most preferably white macula of hypopigmentation, piebaldism, leukoderma, chemical induction by the pigmentation disorder of combination treatment of the present invention.
The melanocytic number that uses in the compositions of the present invention is preferably 1 * 10
3～1 * 10
7Individual cell/cm
2, more preferably 1 * 10
3～1 * 10
6Individual cell/cm
2, again more preferably 1 * 10
4～1 * 10
6Individual cell/cm
2, most preferably 1～5 * 10
5Individual cell/cm
2Scope in.The number of the horn cell that uses in the compositions of the present invention is preferably 1 * 10
4～1 * 10
8Individual cell/cm
2, more preferably 1 * 10
4～1 * 10
7Individual cell/cm
2, again more preferably 1 * 10
5～1 * 10
7Individual cell/cm
2, most preferably about 1 * 10
6Individual cell/cm
2Scope in.In compositions of the present invention, the ratio of melanocyte and horn cell number is preferably 1: 1～and 1: 1000, more preferably 1: 1～1: 100, most preferably be 1: 2～1: 10.
The fibroblastic number that uses in the compositions of the present invention is preferably 1 * 10
3～1 * 10
7Individual cell/cm
2, more preferably 1 * 10
3～1 * 10
6Individual cell/cm
2, again more preferably 1 * 10
4～1 * 10
6Individual cell/cm
2, most preferably about 1 * 10
5Individual cell/cm
2Scope in.In compositions of the present invention, the ratio of melanocyte and fibroblast number is preferably 1: 1～and 1: 1000, more preferably 1: 1～1: 100, most preferably be about 1: 1.
Be used for the present invention's fibroblast unrestricted and preferably comprise people's normal fibroblast, the mitosis non-activity human fibroblasts handled with gamma-rays or mitomycin, animal normal fibroblast, mitosis non-activity animal fibroblast cell or the fibroblast cell line (for example, Swiss 3T3) handled with gamma-rays or mitomycin.Most preferably, be used for fibroblast behaviour normal fibroblast of the present invention.
Pharmaceutical composition of the present invention comprises pharmaceutically suitable carrier and cell mixing preparation.In pharmaceutical preparation, use usually, pharmaceutically suitable carrier comprises lactose, glucose, sucrose, sorbitol, mannitol, starch, acacia gum, potassium phosphate, arginine ester (arginate), gelatin, potassium silicate, microcrystalline Cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxy benzoate, Talcum, magnesium stearate and mineral oil, but is not limited thereto.Can further comprise lubricant, wetting agent, sweetener, flavoring agent, emulsifying agent, suspending agent and antiseptic according to pharmaceutical composition of the present invention.Find among the Remington ' s PharmaceuticalSciences (19th ed., 1995) that the suitable pharmaceutically suitable carrier and the detailed content of preparation can be incorporated herein by reference herein.
Pharmaceutical composition according to the present invention is used for being transplanted to skin.The suitable dosage of pharmaceutical composition of the present invention can change according to drug formulation process, application process, patient age, body weight, sex, pathogenic disease states, diet, time of application, route of administration, excretion rate with to the sensitivity of institute's pharmaceutical composition.The term of using in the literary composition " pharmacy effective dose " refers to the amount that above-mentioned pigmentation disorder is fully showed therapeutic effect.Preferably, pharmaceutical composition of the present invention can every day dosage 10
3～10
9Individual cell is used.
According to routine techniques well known by persons skilled in the art, can prepare with aforesaid pharmaceutically suitable carrier and/or vehicle according to pharmaceutical composition of the present invention, the unit dosage form and the multiple dose form of several forms finally is provided.The limiting examples of described preparation includes, but not limited to suspending agent or Emulsion, extract, elixir, powder, granule, tablet and the capsule in solution, oil or the aqueous medium, and can further comprise dispersant or stabilizing agent.
Pharmaceutical composition of the present invention can be prepared into form of film.For example, from isolating melanocyte of normal skin tissue and horn cell, perhaps melanocyte and fibroblast are cultivated in a thin film altogether, then this film paper are used for affected part.
The preparation of described pharmaceutical composition comprises a kind of be used for using simultaneously melanocyte and horn cell, perhaps melanocyte and fibroblastic preparation, and a kind of final performance identical with application effect simultaneously be used for separate administration melanocyte and horn cell, perhaps melanocyte and fibroblastic preparation.
Cell therapy compositions of the present invention is first by cell therapy scheme treatment pigmentation disorder.Cell mixing preparation or melanocyte that compositions of the present invention comprises melanocyte and horn cell make it induce very effective pigmentation with the treatment disease relevant with hypopigmentation, for example white macula with fibroblastic cell mixing preparation as active component.
Another aspect of the present invention provides a kind of painted skin reconstruction model, described painted skin reconstruction model comprises (i) type i collagen and is embedded in fibroblast in the type i collagen as dermal substitute, (ii) be coated in IV Collagen Type VI and the laminin on the type i collagen and (iii) inoculate and cultivate horn cell and melanocyte on IV Collagen Type VI and laminin.
Painted skin reconstruction model of the present invention is used to replace animal model.
In described painted skin reconstruction model, exist in lowermost portion as the type i collagen of dermal substitute.When preparation dermal substitute layer, relate to fibroblast.Be used for fibroblast of the present invention unrestricted and preferably comprise people's normal fibroblast, the mitosis non-activity human fibroblasts handled with gamma-rays or mitomycin, animal normal fibroblast, mitosis non-activity animal fibroblast cell or the fibroblast cell line (for example, Swiss 3T3) handled with gamma-rays or mitomycin.Most preferably, be used for fibroblast behaviour normal fibroblast of the present invention.Fibroblastic number of inoculation is usually 1 * 10
3～1 * 10
7Individual cell/ml, preferred 1 * 10
4～1 * 10
6Individual cell/ml, most preferably about 1.5 * 10
5In the scope of individual cell/ml.
With IV Collagen Type VI and laminin (basement membrane composition) coating type i collagen adhering to enhancing melanocyte and horn cell.
Melanocyte and horn cell are positioned on the dermal substitute that is coated with IV Collagen Type VI and laminin.Inoculation melanocyte and horn cell and cultivation on dermal substitute.The cultivation of melanocyte and horn cell can be carried out in following one of three conditions being exposed to air that depend on, that is: air drying condition, half-dried condition and submerged conditions.
The term that uses in the literary composition " air drying condition " refers to the horn cell layer is seeded in the type i collagen gel-type vehicle, and promptly epidermis is exposed to airborne condition (referring to Figure 10, A portion) fully.
The term that uses in the literary composition " half-dried condition " refers to the horn cell layer is seeded in the type i collagen gel-type vehicle, i.e. the least possible condition that is covered by substrate (referring to Figure 10, B portion) of epidermis.Under this condition, the factor that influences the horn cell layer works by substrate and air.
The term that uses in the literary composition " submerged conditions " refers to the horn cell layer is seeded in the type i collagen gel-type vehicle, i.e. the condition (referring to Figure 10, C portion) that covered by substrate as far as possible fully of epidermis.Under this condition, the factor that influences the horn cell layer only works by substrate.
The horn cell and the melanocytic cultivation of inoculation are carried out according to two steps.First incubation step (being used to be coated with the cultivation of the type i collagen layer of horn cell) carries out under submerged conditions.The time cycle that is used for first incubation step is preferably 3～10 days, most preferably is 7 days.Second incubation step carries out under air drying condition, and the time cycle be preferably 8～20 days, most preferably be 14 days.
The number of the horn cell of inoculation is usually 1 * 10
3～1 * 10
7Individual cell/cm
2, preferred 1 * 10
4～1 * 10
6Individual cell/cm
2, most preferably about 3 * 10
5Individual cell/cm
2Scope in.The melanocytic number of inoculation is usually 1 * 10
2～1 * 10
6Individual cell/cm
2, preferred 1 * 10
3～1 * 10
5Individual cell/cm
2, most preferably about 3 * 10
4Individual cell/cm
2Scope in.In the skin reconstruction model, the ratio of the number of melanocyte and horn cell is preferably 1: 1～and 1: 1000, more preferably 1: 1～1: 100, most preferably be 1: 2～1: 10.
In the horn cell of in dermal substitute, cultivating and melanocyte, be positioned at low relatively part with basement membrane composition at most external horn cell formation epidermis and melanocyte.Melanocyte in the skin reconstruction model produces melanin and forms to the melanin unit of horn cell migration, thereby successfully rebuilds painted skin.
Reconstruction model of the present invention as be used to verify and study that melanocytic melanocyte generates, melanin to sending of horn cell pass, the model of the effect of the interaction of melanocyte and horn cell and cosmetics (for example, whiteness of skin cosmetics) is very useful.In addition, described reconstruction model can be used as the clinical preceding and zooperal alternative model that is used for skin photoage/photic damage.
Description of drawings
Fig. 1 a-1c represents from the isolated melanocyte of normal human skin tissues, horn cell and fibroblastic immunohistochemical staining result.
Fig. 2 is the curve chart of explanation according to the variation of the melanic amount of isolating melanocytic number.
Fig. 3 is the curve chart of explanation according to the activity change of isolating melanocytic number tryrosinase.
Fig. 4 is transplanted to melanocyte, melanocyte and horn cell or melanocyte and fibroblast for expression the result's of nude mice photo.M: melanocyte, K: horn cell and F: fibroblast.
Fig. 5 is transplanted to melanocyte, horn cell and fibroblastic mixing suspension for expression the photo of 2 week of nude mice back skin color variation.Blended cell suspending liquid comprises 5 * 10
5Individual cell/cm
2Melanocyte, 1 * 10
6Individual cell/cm
2Horn cell and 1 * 10
6Individual cell/cm
2Fibroblast.
Fig. 6 is transplanted to 1 all h and Es (H﹠amp behind the nude mice for expression with melanocyte, horn cell and fibroblastic mixing suspension; E) photo of coloration result.M: melanocyte, K: horn cell and F: fibroblast.The melanocyte major part is found and shows to move to basal layer in corium.
Fig. 7 is transplanted to melanocyte, horn cell and fibroblastic mixing suspension for expression the h and E (H﹠amp in 2 weeks behind the nude mice; E) photo of coloration result.M: melanocyte, K: horn cell and F: fibroblast.Show among the figure that melanocyte moves to basal layer, move to the next door and all be distributed in operation part along basal layer.
Fig. 8 represents that melanin is sent to be delivered to horn cell when melanocyte and horn cell are cultivated altogether.
Fig. 9 represents the result that external painted skin is rebuild.
Figure 10 represents to be used for the melanocyte of external painted skin reconstruction and the cultural method of horn cell.The bottom is a dermal substitute, and the sealing frame table shows that the height of substrate and nail shape represent melanocyte and horn cell.
The specific embodiment
The present invention will further describe by embodiment.These embodiment are used to be described more specifically, and the scope of illustrating in the claims of the present invention be not limited to embodiment or be implemented the example limit, this it will be apparent to those skilled in the art that.
Embodiment
Embodiment 1: melanocyte, horn cell separate and cultivation with fibroblastic
Cultivate from the isolated melanocyte of normal human skin tissues, horn cell and fibroblast and to it.
Embodiment 1-1:
Melanocytic separation and cultivation
(gentamycin, phosphate buffered solution Gibco) (WelGENE) wash 8 times to remove blood and the pollutant that solidify with containing 50 μ g/ml antibiotic with skin histology.Fat deposit in the skin histology corium is removed, and described tissue is cut into the 5mm size, be organized in 4 ℃ and hatched 16 hours with 1mg/ml Bacillus polymyxa Neutral proteinase (Roche) down what cut.Then, isolate epidermis from skin histology, (0.05% trypsin-0.53mM EDTA4Na Gibco) is hatched 30 minutes with trypsin-EDTA solution down with phosphate buffered solution washing epidermis and in 37 ℃.Trypsin-EDTA the solution that will comprise epidermis carries out the several imbibition and is used for from epidermis isolated cell (melanocyte and horn cell).
In comprising the trypsin of isolating melanocyte and horn cell-EDTA solution, add and transfer in the 50ml test tube as the MGM-3 (Cambrex) of melanocyte culture medium and with product, then centrifugal under 300 * g.After the abandoning supernatant, add the melanocyte culture medium and (comprise 10 with the homogenize isolated cells and the solution that will homogenize
7Individual cell) is distributed in the 100mm culture dish.Under 37 ℃ at CO
2Carry out in the incubator cell culture and the next day change culture medium.In case culture converges, the melanocyte culture medium is discarded and culture dish is washed once with phosphate buffered solution.Trypsin-EDTA solution and the described culture dish of 1.5ml were hatched 5 minutes, and by make melanocyte separate true faster from the epidermis cell results melanocyte of cultivating from culture dish than horn cell with trypsinization.The melanocytic cultivation that will comprise collection is centrifugal and abandoning supernatant based on 300 * g.Then, use homogenize suspension melanocyte and of melanocyte culture medium with its aliquot (3 * 10
5Individual cell) transfers to and be used to go down to posterity cultivation in the 100mm culture dish.By using antibody to confirm that cultured cells is melanocyte at melanocytic label HMB-45.
Embodiment 1-2:
The separation of horn cell and cultivation
(gentamycin, phosphate buffered solution Gibco) (WelGENE) wash 8 times to remove blood and the pollutant that solidify with containing 50 μ g/ml antibiotic with skin histology.Fat deposit in the skin histology corium is removed, and described tissue is cut into the 5mm size, with the tissue that cuts with the trypsin solution of the 10ml that adopts the magnetic bar physical stimulation (0.125% trypsin: versene=1: 1, Gibco) under room temperature, hatch 45 minutes with angle of departure cell plastid.In the trypsin solution that comprises isolated cells, the trypsin inhibitor (Gibco) of adding 0.1mg/ml is also transferred in the 50ml test tube, and then 300 * g is centrifugal.After removing supernatant, the KGM (Cambrex) that adds as the horn cell culture medium (comprises 3 * 10 with the homogenize isolated cells and the solution that will homogenize
5Individual cell) is distributed in the 100mm culture dish.Under 37 ℃ at CO
2Carry out in the incubator cell culture and the next day change culture medium.In case culture converges, the horn cell culture medium is discarded and culture dish is washed once with phosphate buffered solution.Trypsin-EDTA solution and the described culture dish of 1.5ml were hatched 10 minutes, and the results horn cell.The cultivation of horn cell that will contain collection is centrifugal and abandoning supernatant based on 300 * g.Then, use homogenize suspension horn cell and of horn cell culture medium with its aliquot (3 * 10
5Individual cell) transfers to and be used to go down to posterity cultivation in the 100mm culture dish.By using antibody to confirm that cultured cells is horn cell at the full cytokeratin of label (pan-cytokeratin) of horn cell.
Embodiment 1-3:
Fibroblastic separation and cultivation
(gentamycin, phosphate buffered solution Gibco) (WelGENE) wash 8 times to remove blood and the pollutant that solidify with containing 50 μ g/ml antibiotic with skin histology.Described tissue is cut into the 5mm size, is organized in 4 ℃ and hatched 16 hours with 1mg/ml Bacillus polymyxa Neutral proteinase (Roche) down what cut.Then, separate epidermis, hatched 2 hours with 1mg/ml collagenase (Roche) down with phosphate buffered solution washing corium and in 37 ℃ from skin histology.In comprising isolating fibroblastic collagenase solution, add as the FGM (Cambrex) of fibroblast culture medium and transfer in the 50ml test tube, then 300 * g is centrifugal.After removing supernatant, add the fibroblast culture medium and (comprise 1 * 10 with the homogenize isolated cells and the solution that will homogenize
5Individual cell) is distributed in the 100mm culture dish.Under 37 ℃ at CO
2Carry out cell culture in the incubator and changed culture medium in per three days.In case culture converges, the melanocyte culture medium is discarded and culture dish is washed once with phosphate buffered solution.Trypsin-EDTA solution and the described culture dish of 1.5ml were hatched 10 minutes, and the results fibroblast.Fibroblastic cultivation that will contain collection is centrifugal and abandoning supernatant based on 300 * g.Then, use homogenize suspension fibroblast and of fibroblast culture medium with its aliquot (1 * 10
5Individual cell) transfers to and be used to go down to posterity cultivation in the 100mm culture dish.By using fibroblastic label Vimentin to confirm that cultured cells is fibroblast.
Embodiment 1-4:
Immunohistochemical staining
Be included in cell in the culture dish with trypsinization to collect all types of cells and to suspend in the culture medium that 1ml is used for each cell type.Cell suspending liquid is distributed to the 24-porous culture dish that comprises coverslip and under 37 ℃ at 5%CO
2Cultivated 24 hours in the incubator, make cell attachment on coverslip: melanocyte, 2 * 10
4Individual cells/well; Horn cell, 1 * 10
4Individual cells/well; And fibroblast 1 * 10
4Individual cells/well.Remove culture medium and wash each hole with 1ml phosphate buffered solution (WelGENE) ,-20 ℃ the methanol that adds 1ml then is to fix 20 minutes under 4 ℃.After discarding methanol, culture dish is hatched the 0.2%Triton X-100 (USB) in the 1ml phosphate buffered solution 10 minutes with each porocyte with the phosphate buffered solution washed twice and under room temperature, then remove 0.2%Triton X-100 and use the phosphate buffered solution washed twice.Hatch each hole 1 hour with the blocking-up non-specific binding with 20% normal goats serum (Sigma) in the 1ml phosphate buffered solution under the room temperature.To in 20% lowlenthal serum, be diluted to 1: 50 at mice-Anti-Human's antibody of each label of cell type and the aliquot of 100 μ l antibody will be added drop-wise on the thin Parafilm: melanocyte, HMB-45 antibody (DAKO); Horn cell, full cytokeratin antibody (DAKO); And fibroblast, Vimentin antibody (DAKO).The careful antibody-solutions that takes out coverslip and its plane contact that is attached with cell is diluted from culture dish.After hatching 1 hour under the room temperature, the coverslip that last plane is had an attached cell is carefully transferred in the culture dish and is washed each hole twice with the 1ml phosphate buffered solution.The bonded goat of FITC--anti--mouse antibodies (Vector) was diluted in phosphate buffered solution 1: 100 and the aliquot of 100 μ l antibody is added drop-wise on the thin Parafilm.Carefully from culture dish take out coverslip and its plane with attached cell is contacted with the antibody-solutions of dilution.After hatching 1 hour under the room temperature, the coverslip that last plane is had an attached cell is carefully transferred in the culture dish and is washed each hole twice with the 1ml phosphate buffered solution.The DAPI (Sigma) of nuclear of will being used for dyeing was diluted to 1: 1000 and added the DAPI solution of 1ml dilution in phosphate buffered solution in each hole, then reaction 5 minutes under the room temperature.With each hole twice of 1ml distilled water wash, and 50 μ l are used for the mounting solution of fluorescence staining, and (Vectashield Vector) is added drop-wise on the microscope slide.From culture dish careful take out coverslip and its plane with attached cell contacted with mounting solution be used under fluorescence microscope, observing.
As shown in Figure 1, isolating melanocyte, horn cell and fibroblast are used HMB45, full cytokeratin and Vimentin antibody staining respectively, and this proves and successfully is separated to melanocyte, horn cell and fibroblast.
Embodiment 2: the analysis of isolating melanocyte titre
To separate among the embodiment 1 and the melanocytic titre of cultivating in order showing, to have measured melanic total amount and the activity that relates to the biosynthetic tryrosinase of melanin in the melanocyte.
The melanic total amount of following analysis: in culture dish, obtain melanocyte and horn cell (negative control) again by trypsinization.Then, in the 50ml test tube, prepare 1 * 10
3Individual cell, 1 * 10
4Individual cell, 2.5 * 10
4Individual cell, 5 * 10
4Individual cell, 7.5 * 10
4Individual cell and 1 * 10
5The melanocyte of individual cell and in the 50ml test tube, prepare 1 * 10
5The horn cell of individual cell.With the cell solution 300 * g of preparation centrifugal with the 1N NaOH solution removing supernatant and in each cell precipitation thing, add 100 μ l with suspension cell.Use the standard solution of 1N NaOH formulations prepared from solutions melanin (Sigma): 1 μ g/ml, 10 μ g/ml, 50 μ g/ml, 100 μ g/ml and 200 μ g/ml.Standard solution and cell preparation with multiple concentration keep being used in 1 hour eluting melanin in 60 ℃ of water-baths.The absorbance that uses spectrophotometric determination 450nm is to analyze melanic total amount.
The activity of following analysis tryrosinase: in culture dish, obtain melanocyte again and (MGM, described melanocyte suspends in Cambrex) in the melanocyte culture medium by trypsinization.Aliquot (2 * 10 with the melanocyte suspension
3Individual cell, 5 * 10
3Individual cell, 1 * 10
4Individual cell and 2 * 10
4Individual cell) transfer to the 96-well culture plate and under 37 ℃ at 5%CO
2Cultivated 24 hours in the incubator.After discarding culture medium, wash in each hole of each Kong Bingxiang the 1%Triton X-100 (USB) in the phosphate buffered solution that adds 90 μ l with 200 μ l phosphate buffered solution.Culture plate is kept down being used for frozen cell in 30 minutes in-70 ℃, then cell is at room temperature melted with the destruction cell membrane, thereby obtain the melanocyte extract.In each hole that comprises the melanocyte extract, add the L-DOPA as the tryrosinase substrate (Sigma) of 10 μ l 10mM and under 37 ℃, hatched 1 hour.When L-DOPA was converted into the brown chemical compound by the tryrosinase in the melanocyte extract, the absorbance that uses spectrophotometric determination 450nm was to analyze the activity of tryrosinase in the melanocyte.
As shown in Figure 2, with the increase of melanocyte number, melanic amount increases.In addition, people's epidermal keratinocyte (HEK) of observing as negative control does not have melanin.
Fig. 3 explanation increases the increased activity of tryrosinase with melanocytic number.
Embodiment 3: melanocyte is transplanted to nude mice
Preparation melanocyte, horn cell and fibroblast suspension.Remove culture medium with being included among the embodiment 1 melanocyte, horn cell or the fibroblastic culture dish that separate with cultivating, with the PBS washing and at 37 ℃ down with trypsin treatment 10 minutes.When cell separates from culture dish bottom, it is suspended in DMEM culture medium (Gibco) and collect, it is centrifugal to follow 300 * g.After the abandoning supernatant, in each cell preparation, add the DMEM culture medium to obtain 5 * 10
5Individual cell/cm
2The melanocyte suspension, 1 * 10
6Individual cell/cm
2Horn cell suspension and 1 * 10
6Individual cell/cm
2The fibroblast suspension.
6 the week age BALB/c SI c-nu/nu male mice (Central Lab.Animal Inc., Korea S) adapted to for 1 week, mixture by peritoneal injection ketamine and xylazine hydrochloride is anaesthetized, and it is used to remove the ethanol disinfection of the back of epidermis with times Temin (betamine) and 70% then.Using pocket knife that the nude mice seat area is removed size is 1 * 1cm
2Epidermis.The DMEM that 15 μ l are inoculated in the zone of removing epidermis as the cell suspending liquid of negative control or 15 μ l as experimental group: experimental group 1,1 * 10
5Individual cell/cm
2Melanocyte; Experimental group 2,1 * 10
5Individual cell/cm
2Melanocyte and 1 * 10
6Individual cell/cm
2Horn cell; Experimental group 3,5 * 10
5Individual cell/cm
2Melanocyte, 1 * 10
6Individual cell/cm
2Horn cell and 1 * 10
6Individual cell/cm
2Fibroblast; And experimental group 4,1 * 10
5Individual cell/cm
2Melanocyte and 1 * 10
6Individual cell/cm
2Fibroblast.Because wound area removed horny layer and had some exudates, cell suspending liquid is only removed the regional residual of epidermis having.The transplanting of horn cell is used to promote melanocytic regeneration of adhering to epidermis, and fibroblastic transplanting is used for the treatment of the corium damage and promotes the formation of basal layer cell and hemi desmosome (join domain of epidermis and basal layer).
Use the Ceresan universal trockenbeize gauze (silicon gauze) (Molnlycke Health Care AB) and Tegaderm (3M) drug of topical application in transplanting the zone.The described silicon gauze that comprises the polyamide matrix that is coated with soft silicon can comprise cell suspending liquid in its hole.Less times greater than the silicon gauze of the wound area skin surface attached to the transplanted cells suspension, and plane surrounds with the wet condition that keeps wound area by Tegaderm and prevents to pollute and dry on it.Continue 5 day to the antibiotic (5mg/ml cefazolin sodium) twice of nude mice peritoneal injection 100 μ l every day.Transplant 1 week or 2 weeks of back, remove the drug of topical application, and analyze pollution, inflammation, recovery and the pigmentation of transplanting the zone from transplanting the zone.The new skin histology that forms in the nude mice is carried out biopsy and uses 10% formalin fixed, then with h and E (H﹠amp; E) dyeing.
The following H﹠amp that carries out; E dyeing: will be embedded in the paraffin tissue slice and attached on the microscope slide, then microscope slide flood 10 minutes to remove tissue paraffin on every side in dimethylbenzene.Then, microscope slide is washed 10 minutes with the hydration tissue and with flowing water washing 10 minutes in proper order with 100%, 90%, 80% and 70% ethanol.(Autohematoxylin ResGen) handles 5 minutes so that nuclear staining washes 10 minutes with water will to organize the usefulness hematoxylin.Then, will organize with Yihong (Sigma) and handle 1 minute, wash 10 minutes with water so that cytoplasm dyes.To organize ethanol sequential processing to be used in 2 minutes dewatering and handling 10 minutes with dimethylbenzene with 70%, 80%, 90% and 100%.(Shandon SyntheticMountant Thermo) handles and loads onto coverslip with a mountant with the tissue on the microscope slide.
In all experimental grouies and contrast, do not observe and pollute and inflammation, almost do not find wound contraction and kept the original size of wound.Think that wound contraction in an embodiment compares the wound contraction much less of finding in the holostrome skin wound of removing corium.
Though nude mice has melanocyte, it does not produce melanin.Therefore, under helping, melanin observes people's melanocyte of transplanting easily.In all experimental grouies and contrast, observe the skin histology regular regeneration.And, compare with only transplanting melanocyte, when transplanting the cell mixing preparation of melanocyte and horn cell, find very a large amount of melanocytes (Fig. 4) at basal layer.Astoundingly, when transplanting melanocyte and fibroblastic cell mixing preparation, at basal layer and the very a large amount of melanocyte (Fig. 4) of hair follicle found around.Should be appreciated that horn cell provides somatomedin and promotes promoting epidermization to melanocyte, thereby assist melanocyte to adhere to.Based on described result, obtain conclusion: the cell mixing preparation of melanocyte and horn cell strengthens the pigmentation again of the skin of forfeiture epidermis.Because fibroblast promotes the formation of basal layer and hemi desmosome (join domain between epidermis and the basal layer), can recognize that it helps melanocyte to be attached to basal layer.
Transplant the back perusal of 2 weeks and find, because people's melanocyte of transplanting, the skin (Fig. 5) that forms brown is induced in the transplanting that comprises melanocyte, horn cell and fibroblastic mixed cellularity group.As the painted result of h and E, though its demonstration has regenerated skin and WD corium, only the negative control of injection of culture medium shows does not have melanocyte.On the contrary, comprise in the experimental group 3 of melanocyte, horn cell and fibroblastic mixed cellularity group, transplant the main melanocyte that discovery is transplanted in corium in 1 week back, and produce aixs cylinder melanin is delivered to horny layer (Fig. 6) in transplanting.After transplanting for 2 weeks, the melanocyte that is positioned at corium is moved to basal layer also actively melanin is delivered to horn cell (Fig. 7) at whole basal layer performance uniform distribution.These results show that the melanocyte that exists in the corium is moved upwards up to basal layer in time, along the successive again coloring process of basement membrane migration with the horn cell around in whole basal layer distribution and by melanocytic aixs cylinder melanin being delivered to.The melanin unit of finding in this structure relevant with pigmentation again and the application on human skin is identical, shows that the melanocyte of being transplanted to mice causes the formation as the painted skin of normal human skin.
Therefore, be appreciated that from having separated and at In vitro culture as the melanocyte of the patient's of the pigmentation disorder of white macula normal skin, with mix to obtain blended cell suspending liquid as horn cell and fibroblastic other Skin Cells, then it is administered to affected part, pigmentation is effective again.Animal model in an embodiment is considered to be used to confirm the preclinical models of the effect of the interaction of melanocyte generation, melanocyte and horn cell or whiteness of skin or UV blocking-up cosmetics.
Embodiment 4: the reconstruction of painted skin
The main cause of skin epithelial cell differentiation is calcium and air drying condition.Carry out dimensional culture by on the corium of forming by collagen and fibroblast, cultivating epithelial cell.Epithelial cell is cultivated to be divided into keratinized stratified squamous epithelium in interface between air layer and water layer, and melanocyte produces melanin and melanin is delivered to horn cell at basal layer, thereby forms the melanin unit to comprise the melanocytic skin texture similar to application on human skin.
The preparation collagen mixture is as dermal substitute.By with 8: 1: 1 ratio mixing type i collagen (Nitta), 10 * E-culture medium (comprise DMEM: F12=3: 1) and reconstitution buffers (contain 2.2g NaHCO in the 0.05N NaOH solution of 100ml
3With 4.77g HEPES) the preparation collagen mixture.The preparation of carrying out dermal substitute under 4 ℃ is to prevent the gelation of collagen.In the 24-orifice plate, prepare 0.6cm
2Plug-in type Tissue Culture Dish (Millicell) (Millipore, Inc).From culture dish, obtain fibroblast again and with 1.5 * 10
5The concentration of individual cell/ml suspends in collagen mixture.In this step, at first fibroblast is suspended in culture medium, the volume of adjusting culture medium be no more than collagen mixture culture volume 10%, in collagen mixture, suspend then.200 μ l collagen mixtures are poured into collagen mixture was solidified 30 minutes～1 hour in 37 ℃ of incubators.The E-culture medium that adds 200 μ l in collagen mixture (comprises DMEM: F12=3: 1) and to the outside of plug-in type Tissue Culture Dish add culture medium so that plug-in type Tissue Culture Dish inside and outside has identical culture medium height.Cultivate after 24 hours, with top the adhering to of IV Collagen Type VI and laminin coating dermal substitute with promotion melanocyte and horn cell.Use the 0.2mg/ml collagen of 20 μ l and the mixed solution of 30 μ g/ml laminins under 37 ℃, to apply 1 hour.
Then, with horn cell and melanocyte with trypsinization obtaining again from culture dish and to mix with 1: 10 ratio, then with it respectively with 3 * 10
5Individual cell/cm
2With 3 * 10
4Individual cell/cm
2Concentration be inoculated into simultaneously on the collagen corium of coating.In 4 hours, horn cell and melanocyte are attached to the top of dermal substitute.Per two days or per three days are changed culture medium and were cultivated 7 days.When horn cell and melanocyte are evenly grown on the top of dermal substitute, in order to cultivate epidermis in the interface between water and air, remove supracutaneous culture medium, and add culture medium, under air drying condition, cultivated 14 days to the outside of plug-in type Tissue Culture Dish.
Shown in Fig. 8 and 9, after 14 days, inoculation horn cell and melanocyte all cause forming in the skin reconstruction model with the painted skin of melanin in air-dry cultivation.Yet, in only using the skin reconstruction model of horn cell, do not observe this pigmentation.And, when the skin reconstruction model is cut into slices and dyeed, find that the horn cell differentiation is to form as the keratinized skin of application on human skin and produce and send melanin to the horn cell that breaks up attached to the melanocyte of the basal layer of keratinized skin.
Observe the melanocyte that is positioned at basal layer and generation and send melanic melanin unit to the horn cell layer.Therefore, these results impel us inference can comprise melanocytic application on human skin spline structure and replace animal model in external structure.
As indicated above, the invention provides a kind of pharmaceutical composition that is used for the cell therapy of pigmentation disorder.In addition, the invention provides a kind of painted skin reconstruction model.Compositions of the present invention is used for first by cell therapy scheme treatment pigmentation disorder.Cell mixing preparation or melanocyte that compositions of the present invention comprises melanocyte and horn cell make it can induce pigmentation with the hypopigmentation relevant disease of treatment as white macula very effectively with fibroblastic cell mixing preparation as active component.
Described embodiment preferred of the present invention, should be appreciated that the variation and the change that fall into essence of the present invention it will be apparent to those skilled in the art that, and scope of the present invention is determined by claims and equivalent thereof.
Claims (23)
1, a kind of pharmaceutical composition that is used for the cell therapy of pigmentation disorder, this pharmaceutical composition comprise (a) melanocyte and horn cell or (b) melanocyte and fibroblast as active component.
2, pharmaceutical composition according to claim 1, wherein, described melanocyte, horn cell and/or fibroblast are isolated from skin, perhaps isolate and at In vitro culture from skin.
3, pharmaceutical composition according to claim 1, wherein, described pigmentation disorder is leukoderma, pityriasis alba, nevus depigmentation, postinflammatory depigmentation or the white macula of hypopigmentation, piebaldism, leukoderma, chemical induction.
4, pharmaceutical composition according to claim 1, wherein, described melanocytic number is 1 * 10
3～1 * 10
6Individual cell/cm
2, the number of described horn cell is 1 * 10
4～1 * 10
7Individual cell/cm
2
5, pharmaceutical composition according to claim 1, wherein, described fibroblastic number is 1 * 10
3～1 * 10
6Individual cell/cm
2
6, pharmaceutical composition according to claim 1, wherein, the active component of described compositions comprises melanocyte, horn cell and fibroblast.
7, a kind of painted skin reconstruction model, this painted skin reconstruction model comprises (i) type i collagen and is embedded in fibroblast in the type i collagen as dermal substitute, (ii) be coated in IV Collagen Type VI on the type i collagen and laminin and (iii) inoculation and horn cell and the melanocyte cultivated on IV Collagen Type VI and laminin.
8, painted skin reconstruction model according to claim 7, wherein, described horn cell and melanocyte are cultivated under air drying condition.
9, painted skin reconstruction model according to claim 7, wherein, described horn cell forms the horn cell layer.
10, painted skin reconstruction model according to claim 9, wherein, described melanocyte produces the melanin to the migration of horn cell layer.
11, painted skin reconstruction model according to claim 7, wherein, the ratio of described melanocyte and horn cell number is 1: 2～1: 10.
12, a kind of method for the treatment of pigmentation disorder, described method comprise use to the experimenter (i) melanocyte that comprises (a) pharmacy effective dose and horn cell or (ii) melanocyte and fibroblast as active component and (b) pharmaceutical composition of pharmaceutically suitable carrier.
13, method according to claim 12, wherein, described melanocyte, horn cell and/or fibroblast are isolated from skin, perhaps isolate and at In vitro culture from skin.
14, method according to claim 12, wherein, described pigmentation disorder is leukoderma, pityriasis alba, nevus depigmentation, postinflammatory depigmentation or the white macula of hypopigmentation, piebaldism, leukoderma, chemical induction.
15, method according to claim 12, wherein, described melanocytic number is 1 * 10
3～1 * 10
6Individual cell/cm
2, the number of described horn cell is 1 * 10
4～1 * 10
7Individual cell/cm
2
16, method according to claim 12, wherein, described fibroblastic number is 1 * 10
3～1 * 10
6Individual cell/cm
2
17, method according to claim 12, wherein, described pharmaceutical composition comprises melanocyte, horn cell and fibroblast.
18, comprise (i) melanocyte and horn cell or (ii) melanocyte and fibroblastic cell mixture are used to prepare the purposes of the medicine for the treatment of pigmentation disorder.
19, purposes according to claim 18, wherein, described melanocyte, horn cell and/or fibroblast are isolated from skin, perhaps isolate and at In vitro culture from skin.
20, purposes according to claim 18, wherein, described pigmentation disorder is leukoderma, pityriasis alba, nevus depigmentation, postinflammatory depigmentation or the white macula of hypopigmentation, piebaldism, leukoderma, chemical induction.
21, purposes according to claim 18, wherein, described melanocytic number is 1 * 10
3～1 * 10
6Individual cell/cm
2, the number of described horn cell is 1 * 10
4～1 * 10
7Individual cell/cm
2
22, purposes according to claim 18, wherein, described fibroblastic number is 1 * 10
3～1 * 10
6Individual cell/cm
2
23, purposes according to claim 18, wherein, described cell mixture comprises melanocyte, horn cell and fibroblast.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310231829.8A CN103356701B (en) | 2005-11-25 | 2006-06-20 | Pharmaceutical composition for cell therapy of pigmentation disorders |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020050113287 | 2005-11-25 | ||
| KR20050113287 | 2005-11-25 |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310231829.8A Division CN103356701B (en) | 2005-11-25 | 2006-06-20 | Pharmaceutical composition for cell therapy of pigmentation disorders |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101351217A true CN101351217A (en) | 2009-01-21 |
Family
ID=38067372
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2006800503254A Pending CN101351217A (en) | 2005-11-25 | 2006-06-20 | Pharmaceutical compositions for cell therapy of pigmentation disorders |
| CN201310231829.8A Expired - Fee Related CN103356701B (en) | 2005-11-25 | 2006-06-20 | Pharmaceutical composition for cell therapy of pigmentation disorders |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310231829.8A Expired - Fee Related CN103356701B (en) | 2005-11-25 | 2006-06-20 | Pharmaceutical composition for cell therapy of pigmentation disorders |
Country Status (3)
| Country | Link |
|---|---|
| KR (1) | KR100806695B1 (en) |
| CN (2) | CN101351217A (en) |
| WO (1) | WO2007061168A1 (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104877957A (en) * | 2014-02-28 | 2015-09-02 | 上海尚瑞生物医药科技有限公司 | Cell strain for promoting pigment generation of melanocytes |
| CN105112353A (en) * | 2015-07-29 | 2015-12-02 | 赫柏慧康生物科技无锡有限公司 | Mixed cultivation method of keratinocyte and melanocyte and application |
| CN106999372A (en) * | 2014-09-29 | 2017-08-01 | Elc 管理有限责任公司 | For stimulating the differentiation of keratinocyte with the active matter of the hyperpigmented skin of whitening |
| CN107683148A (en) * | 2015-03-26 | 2018-02-09 | 波尔多大学 | Skin renewal method |
| CN109475666A (en) * | 2016-07-29 | 2019-03-15 | 欧莱雅 | Skin equivalent with dermal compartment distinguishing, arranged side by side |
| CN110257327A (en) * | 2019-06-27 | 2019-09-20 | 重庆市细胞生物工程技术有限公司 | A kind of isolated culture method of umbilical cord mesenchymal stem cells |
| CN113166694A (en) * | 2018-11-30 | 2021-07-23 | 株式会社资生堂 | Pigmentation skin model, method for producing same, and method for evaluating factor for treating or preventing skin pigmentation |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2658074C (en) | 2008-03-17 | 2017-11-07 | L'oreal | Functional pigmented skin equivalent |
| FR2928654B1 (en) * | 2008-03-17 | 2018-04-27 | L'oreal | EQUIVALENT OF FUNCTIONAL PIGMENTED SKIN. |
| KR101328561B1 (en) * | 2010-05-11 | 2013-11-13 | (주) 메디컬그룹베스티안 | Method for preparaing autologous agent for alleviation or treatment of skin defects |
| KR101431883B1 (en) * | 2011-11-24 | 2014-08-21 | 경희대학교 산학협력단 | A co-culture system of 3-dimensional artificial tissue and monitering cells to substitute for animal test, and manufacturing method thereof |
| CN103983762A (en) * | 2013-02-08 | 2014-08-13 | 北京富龙康泰生物技术有限公司 | Melanocyte-containing skin model, construction method and application thereof |
| EP3274445A1 (en) * | 2015-03-26 | 2018-01-31 | Université de Bordeaux | Skin equivalent and use |
| EP3336175B1 (en) * | 2016-12-16 | 2023-08-30 | Universite De Bordeaux | Three-dimensional model of depigmenting disorder |
| SG11202105105SA (en) | 2018-11-30 | 2021-06-29 | Shiseido Co Ltd | Composition for treating or preventing skin pigmentation |
| CN114686420B (en) * | 2022-04-08 | 2023-11-28 | 济南磐升生物技术有限公司 | Preparation method and application of 3D epidermis model |
-
2006
- 2006-04-25 KR KR1020060037022A patent/KR100806695B1/en active IP Right Grant
- 2006-06-20 WO PCT/KR2006/002366 patent/WO2007061168A1/en active Application Filing
- 2006-06-20 CN CNA2006800503254A patent/CN101351217A/en active Pending
- 2006-06-20 CN CN201310231829.8A patent/CN103356701B/en not_active Expired - Fee Related
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104877957A (en) * | 2014-02-28 | 2015-09-02 | 上海尚瑞生物医药科技有限公司 | Cell strain for promoting pigment generation of melanocytes |
| CN104877957B (en) * | 2014-02-28 | 2018-06-01 | 上海尚瑞生物医药科技有限公司 | A kind of cell line that melanocyte pigment is promoted to generate |
| CN106999372A (en) * | 2014-09-29 | 2017-08-01 | Elc 管理有限责任公司 | For stimulating the differentiation of keratinocyte with the active matter of the hyperpigmented skin of whitening |
| CN107683148A (en) * | 2015-03-26 | 2018-02-09 | 波尔多大学 | Skin renewal method |
| CN107683148B (en) * | 2015-03-26 | 2021-05-04 | 波尔多大学 | Skin reconstruction method |
| CN105112353A (en) * | 2015-07-29 | 2015-12-02 | 赫柏慧康生物科技无锡有限公司 | Mixed cultivation method of keratinocyte and melanocyte and application |
| CN109475666A (en) * | 2016-07-29 | 2019-03-15 | 欧莱雅 | Skin equivalent with dermal compartment distinguishing, arranged side by side |
| CN109475666B (en) * | 2016-07-29 | 2021-08-10 | 欧莱雅 | Skin equivalent with distinct, juxtaposed dermal compartments |
| CN113166694A (en) * | 2018-11-30 | 2021-07-23 | 株式会社资生堂 | Pigmentation skin model, method for producing same, and method for evaluating factor for treating or preventing skin pigmentation |
| CN110257327A (en) * | 2019-06-27 | 2019-09-20 | 重庆市细胞生物工程技术有限公司 | A kind of isolated culture method of umbilical cord mesenchymal stem cells |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2007061168A1 (en) | 2007-05-31 |
| CN103356701A (en) | 2013-10-23 |
| KR20070055309A (en) | 2007-05-30 |
| CN103356701B (en) | 2016-08-31 |
| KR100806695B1 (en) | 2008-02-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101351217A (en) | Pharmaceutical compositions for cell therapy of pigmentation disorders | |
| TWI278517B (en) | Methods and compositions for the differentiation of human preadipocytes into adipocytes | |
| Michel et al. | Characterization of a new tissue-engineered human skin equivalent with hair | |
| Reynolds et al. | Cultured dermal papilla cells induce follicle formation and hair growth by transdifferentiation of an adult epidermis | |
| CN102027106B (en) | Methods for producing hair microfollicles and de novo papillae and their use for in vitro tests and in vivo implantations | |
| ES2252259T3 (en) | THREE-DIMENSIONAL SKIN MODEL. | |
| CN101352586B (en) | Method for preparing full-thickness skin for toxicity test by stem cell raft type cultivation | |
| JP2022068312A (en) | Tissue culture apparatus and method | |
| KR102426746B1 (en) | Method and system for regenerating hair using 3D organoid system of hair follicle stem cells | |
| PT91676A (en) | THREE-DIMENSIONAL CELL AND TISSUE CULTURE SYSTEM | |
| CN107106613A (en) | For the composition for including source of human stem cell excretion body into fat induction, adipose tissue regeneration, skin-whitening or improvement wrinkle | |
| Kinikoglu et al. | Reconstruction of a full-thickness collagen-based human oral mucosal equivalent | |
| CN104548209B (en) | Tissue-engineered epidermis and preparation method thereof | |
| CN106420390A (en) | Stem cell preparation for skin beauty and preparation method thereof | |
| CN101318030A (en) | Method for preparing organ type artificial skin for toxicity inspection by employing built-in cultivation method | |
| US20170252377A1 (en) | Compositions and methods for producing reconstituted skin | |
| EP2737055B1 (en) | Micro organ comprising mesenchymal and epithelial cells | |
| JP5892576B2 (en) | Method for producing epithelial reconstructed body and screening method using the epithelial reconstructed body | |
| Kalyanaraman et al. | Wound healing on athymic mice with engineered skin substitutes fabricated with keratinocytes harvested from an automated bioreactor | |
| Kim et al. | Effects of Cervi cornus Colla (deer antler glue) in the reconstruction of a skin equivalent model | |
| WO2023058429A1 (en) | Method for producing cell aggregate having hair regeneration ability, and method related thereto | |
| CN103764816B (en) | The method for rebuilding scalp model and screening bioactive molecule | |
| Lei et al. | Bioengineered skin substitutes | |
| KR20070108846A (en) | Pharmaceutical compositions for cell therapy of pigmentation disorders | |
| CN111117945B (en) | Skin model containing melanin, construction method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20090121 |