WO2006005977B1 - Novel method for culturing keratinocytes, melanocytes and fibroblasts for skin grafting - Google Patents
Novel method for culturing keratinocytes, melanocytes and fibroblasts for skin graftingInfo
- Publication number
- WO2006005977B1 WO2006005977B1 PCT/HU2005/000076 HU2005000076W WO2006005977B1 WO 2006005977 B1 WO2006005977 B1 WO 2006005977B1 HU 2005000076 W HU2005000076 W HU 2005000076W WO 2006005977 B1 WO2006005977 B1 WO 2006005977B1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cell
- cultured
- melanocytes
- keratinocytes
- hairy scalp
- Prior art date
Links
- 210000002950 fibroblast Anatomy 0.000 title claims abstract 20
- 210000002510 keratinocyte Anatomy 0.000 title claims abstract 17
- 210000002752 melanocyte Anatomy 0.000 title claims abstract 17
- 238000000034 method Methods 0.000 title claims abstract 17
- 210000003491 skin Anatomy 0.000 title claims abstract 8
- 238000012258 culturing Methods 0.000 title claims 5
- 238000002360 preparation method Methods 0.000 claims abstract 10
- 210000004027 cell Anatomy 0.000 claims abstract 8
- 238000004113 cell culture Methods 0.000 claims abstract 5
- 210000004761 scalp Anatomy 0.000 claims 15
- 210000001339 epidermal cell Anatomy 0.000 claims 11
- 210000004748 cultured cell Anatomy 0.000 claims 6
- 230000002500 effect on skin Effects 0.000 claims 6
- 210000004927 skin cell Anatomy 0.000 claims 6
- 238000002054 transplantation Methods 0.000 claims 6
- 239000006285 cell suspension Substances 0.000 claims 5
- 239000002609 medium Substances 0.000 claims 5
- 239000006228 supernatant Substances 0.000 claims 3
- 239000003636 conditioned culture medium Substances 0.000 claims 2
- 239000006143 cell culture medium Substances 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 210000003527 eukaryotic cell Anatomy 0.000 abstract 3
- 206010052428 Wound Diseases 0.000 abstract 2
- 208000027418 Wounds and injury Diseases 0.000 abstract 2
- 230000007547 defect Effects 0.000 abstract 2
- 210000001519 tissue Anatomy 0.000 abstract 2
- 208000032544 Cicatrix Diseases 0.000 abstract 1
- 102000009123 Fibrin Human genes 0.000 abstract 1
- 108010073385 Fibrin Proteins 0.000 abstract 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 abstract 1
- 208000025865 Ulcer Diseases 0.000 abstract 1
- 206010047642 Vitiligo Diseases 0.000 abstract 1
- 230000001684 chronic effect Effects 0.000 abstract 1
- 239000000470 constituent Substances 0.000 abstract 1
- 201000010099 disease Diseases 0.000 abstract 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract 1
- 229950003499 fibrin Drugs 0.000 abstract 1
- 201000009442 piebaldism Diseases 0.000 abstract 1
- 231100000241 scar Toxicity 0.000 abstract 1
- 230000037387 scars Effects 0.000 abstract 1
- 239000000725 suspension Substances 0.000 abstract 1
- 208000024891 symptom Diseases 0.000 abstract 1
- 231100000397 ulcer Toxicity 0.000 abstract 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3886—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells comprising two or more cell types
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3813—Epithelial cells, e.g. keratinocytes, urothelial cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/60—Materials for use in artificial skin
-
- 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
- C12N5/0602—Vertebrate cells
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
- C12N5/0626—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
- 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
- C12N5/0602—Vertebrate cells
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
- C12N5/0629—Keratinocytes; Whole skin
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/70—Undefined extracts
- C12N2500/80—Undefined extracts from animals
- C12N2500/84—Undefined extracts from animals from mammals
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Dermatology (AREA)
- Cell Biology (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Transplantation (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Medicinal Chemistry (AREA)
- Botany (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Urology & Nephrology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Materials For Medical Uses (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention in general relates to the field of eukaryotic cell culture and tissue engineering, more closely to a method for the cultivation of eukaryotic cells and the use of cells cultured according to this method for the preparation of cell transplants e.g. autologous cell transplants. In particular, the invention relates to the use of keratinocyte and/or melanocyte and/or fibroblast transplants prepared according to the present invention for the treatment of various dermatological symptoms and diseases. The invention further relates to keratinocyte and/or melanocyte and/or fibroblast transplants, e.g. cell culture preparations, prepared according to the present invention. The invention concerns areas involving eukaryotic cell culture and tissue engineering, particularly fibrin-containing keratinocyte, melanocyte and fibroblast suspensions based on three constituents. The invention is particularly useful in the treatment of various conditions of skin, e.g. chronic ulcers of different origins, burn wounds, post-traumatic wound defects, defects of extensive, benign, naevoid skin changes and of scars after prior dermabrasion, vitiligo, piebaldism, etc.
Claims
AMENDED CLAIMS received by the International Bureau on 29 December 2005 (29.12.2005)
1 Method for autologous skin cell transplantation, comprising the steps of i) obtaining hairy scalp of a subject having an affected skin area, said hairy scalp comprising* at least partly the bulge, ii) obtaining an epidermal cell suspension from the said hairy scalp. iii) separating epidermal cells from other cell types of the hairy scalp, at least from dermal fibroblasts, and optionally also separating dermal fibroblasts flroro the hairy scalp, « iii) culturing the said epidermal cells in a suitable medium, iv) administering the cultured epidermal cells to the affected skin area,
2. The method of claim 1 wherein epidermal cells comprise keratinocytes and/or melanocytes.
3. The method of claim 2 wherein keratinocytes and melanocytes axe separated from each other and cultured separately in pure cultures.
4. The method of claim 3 wherein the keratinocyte and melanocyte cultures are reconstituted later in desired ratios.
5. The method of any of claims 1 to 4 wherein fibroblasts are also separated from the hairy scalp and cultured separately, and at least one of the following steps is carried out: a) supernatant of the fibroblast culture or a portion thereof is added to the epidermal cell culture ox the culture of keratinocytes and/or melanocytes, b) cultured fibroblasts are administered to the affected skin area, in a desired ratio relative to the other cells,
6. Method for the manufacture of a cultured cell preparation for skin cell transplantation comprising the steps of i) obtaining hairy scalp of a subjects said hairy scalp comprising, at least partly the bulge, ii) obtaining epidermal cells from the said hairy scalp, iii) separating epidermal cells from other cell types of the hairy scalp, at least from dermal fibroblasts, and optionally also separating dermal fibroblasts from the hairy scalp, iv) culturing the said epidermal cells in a suitable medium, thereby obtaining a cell suspension, v) formulating said cell suspension to a cultured cell preparation for skin cell transplantation to an affected skin area.
7. The method of claim 6 wherein epidermal cells comprise keratinocytes and/or melanocytes.
8< The method of claim 7 wherein both, keratinocytes and melanocytes axe used and cultured together in a mixed culture, in a medium providing suitable conditions for both cell types.
9 The method of claim 7 wherein keratinocytes and melanocytes are separated from each other and cultured separately.
10 The method of claim 9 wherein keratinocyte and melanocyte cultures are reconstituted in a pre-determined ratio to obtain a cell suspension comprising a desired ratio of both keratinocytes and melanocytes and this cell suspension is formulated to a cultured cell preparation for skin cell transplantation.
11. The method of any of claims 6 to 10 wherein fibroblasts are also separated from the hairy scalp and cultured separately, and at least one of the following steps is carried out: a) supernatant of the fibroblast culture or a portion thereof is added to the epidermal cell culture or the culture of keratinocytes and/or melanocytes, b) cultured fibroblasts are formulated to a cultured cell preparation for skin cell transplantation and optionally added to keratinocyte and melanocyte culture(s) in a predetermined ratio.
12. A cultured cell preparation or a cultured cell preparation kit for skin cell transplantation in a subject, said cell preparation or kit comprising cultured melanocytes and/or keratinocytes and/or fibroblasts and being obtainable by a method of any of claims 6 to 11.
13. Method for the preparation of a fibroblast conditioned medium, comprising the steps of: i) obtaining hairy scalp of a subject having an affected skin area, said hairy scalp comprising, at least partly the bulge, ii) separating dermal fibroblasts from the hairy scalp, iii) culturing the said dermal fibroblasts separately in a suitable medium, iv) supernatant of the fibroblast culture or a portion thereof is obtained.
14. A cell culture medium for culturing keratinocytes and/or melanocytes, said medium comprising fibroblast conditioned medium obtainable by the method of claim 13.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP05763133A EP1768716A1 (en) | 2004-07-09 | 2005-07-11 | Novel method for culturing keratinocytes, melanocytes and fibroblasts for skin grafting |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| HUP0401385 | 2004-07-09 | ||
| HU0401385A HU227723B1 (en) | 2004-07-09 | 2004-07-09 | Autologous keratinocytes, melanocytes and fibroblast culturing technique and serum-free medium for use in human therapy |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| WO2006005977A1 WO2006005977A1 (en) | 2006-01-19 |
| WO2006005977B1 true WO2006005977B1 (en) | 2008-12-24 |
Family
ID=89985359
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/HU2005/000076 WO2006005977A1 (en) | 2004-07-09 | 2005-07-11 | Novel method for culturing keratinocytes, melanocytes and fibroblasts for skin grafting |
Country Status (3)
| Country | Link |
|---|---|
| EP (1) | EP1768716A1 (en) |
| HU (1) | HU227723B1 (en) |
| WO (1) | WO2006005977A1 (en) |
Families Citing this family (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100303770A1 (en) * | 2006-12-28 | 2010-12-02 | John Maslowski | Methods for culturing dermal cells for treatment of skin injuries such as burns |
| DE102007049402A1 (en) | 2007-10-15 | 2009-04-16 | Euroderm Gmbh | Cosmetic method for increasing the pigmentation of skin using melanocyte progenitor cells |
| EP2198887A4 (en) * | 2007-10-19 | 2011-11-30 | Univ Tokyo | Therapeutic agent for leukoderma and method for acceleration of pigmentation |
| GB2483622A (en) * | 2009-08-03 | 2012-03-21 | Univ Durham | Fibroblast feeder cells |
| GB0916370D0 (en) * | 2009-09-18 | 2009-10-28 | Avecia Biolog Ltd | Compositions |
| CN102858955A (en) * | 2010-02-23 | 2013-01-02 | 国家医疗保健研究所 | Methods for preparing human melanocytes from human pluripotent stem cells |
| DE102010009571A1 (en) * | 2010-02-26 | 2011-09-01 | Euroderm Gmbh | A method of promoting the restoration of bodily function using cells of the hair root sheath, preparation and manufacturing method |
| DE102010009572A1 (en) * | 2010-02-26 | 2011-09-01 | Euroderm Gmbh | A method of promoting healing of skin wounds using the hair root sheath cells, preparation and method of manufacture |
| US9222076B2 (en) * | 2010-05-21 | 2015-12-29 | Rodrigo Focion Soto Pareja | Process for the production of patches or derssings of autologous skin through cultivation of autologous keratinocytes and fibroblasts with autologous serum for the generation of skin |
| JP6101066B2 (en) * | 2012-12-10 | 2017-03-22 | 日本メナード化粧品株式会社 | Preparation method of melanocyte population |
| CN107151648B (en) * | 2017-06-18 | 2023-07-21 | 广东博溪生物科技有限公司 | Culture medium for co-culture of melanocytes and keratinocytes |
| KR102130599B1 (en) * | 2017-12-06 | 2020-07-07 | 주식회사 이뮤노맥스 | Method for Generation of Cytotoxic T Lymphocytes and Use Thereof |
| CN114522182A (en) * | 2020-11-07 | 2022-05-24 | 海口仁术皮肤科门诊部有限公司 | Preparation method of hair follicle melanin stem cell suspension for treating leucoderma |
| CN115227876B (en) * | 2022-07-28 | 2023-08-29 | 福建省海西细胞生物工程有限公司 | Preparation method of tissue engineering epidermis |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19958693C2 (en) * | 1999-12-06 | 2002-07-18 | Biotissue Technologies Gmbh | Methods and compositions for making cell grafts |
-
2004
- 2004-07-09 HU HU0401385A patent/HU227723B1/en not_active IP Right Cessation
-
2005
- 2005-07-11 EP EP05763133A patent/EP1768716A1/en not_active Withdrawn
- 2005-07-11 WO PCT/HU2005/000076 patent/WO2006005977A1/en active Application Filing
Also Published As
| Publication number | Publication date |
|---|---|
| WO2006005977A1 (en) | 2006-01-19 |
| HU0401385D0 (en) | 2004-09-28 |
| HUP0401385A2 (en) | 2006-09-28 |
| EP1768716A1 (en) | 2007-04-04 |
| HU227723B1 (en) | 2012-01-30 |
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