WO2007061205A1 - Procede de culture de fibroblastes faisant appel a un extrait placentaire et composition de regeneration de la peau associee - Google Patents

Procede de culture de fibroblastes faisant appel a un extrait placentaire et composition de regeneration de la peau associee Download PDF

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Publication number
WO2007061205A1
WO2007061205A1 PCT/KR2006/004880 KR2006004880W WO2007061205A1 WO 2007061205 A1 WO2007061205 A1 WO 2007061205A1 KR 2006004880 W KR2006004880 W KR 2006004880W WO 2007061205 A1 WO2007061205 A1 WO 2007061205A1
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Prior art keywords
fibroblasts
culturing
placenta extract
serum
cultured
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PCT/KR2006/004880
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English (en)
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Jun Ho Shin
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Jun Ho Shin
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Priority claimed from KR1020060106452A external-priority patent/KR100701297B1/ko
Application filed by Jun Ho Shin filed Critical Jun Ho Shin
Publication of WO2007061205A1 publication Critical patent/WO2007061205A1/fr

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    • 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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0656—Adult fibroblasts
    • 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
    • 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
    • A61L27/3895—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 using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9728—Fungi, e.g. yeasts
    • 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
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/06—Flowable or injectable implant compositions
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components

Definitions

  • the present invention relates to a method of culturing fibroblasts and a skin- regenerating composition comprising the cultured fibroblasts. More particularly, the present invention relates to a method of culturing fibroblasts using a placenta extract, such that a large amount of fibroblasts are cultured in a short time, and the cultured fibroblasts contain no cells other than fibroblasts and no medium serum-derived proteins, and thus do not cause an immune response when they are injected into the skin, as well as a skin-regenerating composition comprising the cultured fibroblasts.
  • Collagen is a fibrous protein founded between cells in the body of animals, including human beings. Such collagen has been extracted from the skin and bone of livestock (particularly, bovine). Collagen derived from animal has no problem when it is used for edible purposes, but it has a problem of causing an immune response when it is used as an injectable composition.
  • human collagen is used.
  • Human collagen also requires crosslinking or other chemical treatments, like bovine collagen.
  • injectable human collagen obtained from the tissue of a subject is commercially available under the registered trademark "AUTOLOGEN”.
  • the human collagen also does not provide a long-term therapeutic effect, like bovine collagen, is limited only to patients who undergone surgery, and still has the problem of causing an immune response.
  • Korean Patent Registration No. 458268 discloses a method of preparing collagen by culturing human fibroblasts.
  • this method has shortcomings in that the process of culturing fibroblasts is complicated and time- consuming, and collagen should be extracted for use in this method.
  • US Patent Publication No. 2004/0101959 discloses a method of regenerating the skin using autologous fibroblasts having excellent histocompatibility.
  • this method has a problem in that much time is taken for the regeneration of fibroblasts.
  • the present inventors have developed a method of culturing a large amount of collagen-producing fibroblasts in a short time using a placenta extract.
  • a composition containing the fibroblasts cultured using this method can promote skin regeneration in a convenient manner without side effects, when it is injected into the skin.
  • Another object of the present invention is to provide a method of culturing fibroblasts using both a placenta extract and bovine fetal serum.
  • Still another object of the present invention is to provide a method capable of culturing a large amount of fibroblasts in a short time.
  • Yet another object of the present invention is to provide a method of culturing fibroblasts, which do not contain cells other than fibroblasts and no medium serum- derived proteins.
  • Still another object of the present invention is to provide a method of culturing fibroblasts using autologous fibroblasts.
  • Another further object of the present invention is to provide a skin- regenerating composition containing said cultured fibroblasts.
  • the present invention provides a method for culturing fibroblasts, comprising the steps of: (A) preparing fibroblasts; (B) culturing the fibroblasts using a culture medium containing a placenta extract; (C) incubating the cultured fibroblasts in a serum-free medium; and (D) isolating the incubated fibroblasts, wherein the isolated fibroblasts contains no cells other than fibroblasts and no medium serum-derived proteins.
  • the fibroblasts in the step (A) can be autologous fibroblasts.
  • the step of culturing the fibroblasts can be carried out using both the placenta extract and bovine fetal serum.
  • the concentration of the placenta extract used in the step (B) of culturing the fibroblasts is 0.05-20%
  • the step (C) of incubating the cultured fibroblasts in the serum-free medium can be carried out at a temperature of 30-40 °C for 10-20 hours.
  • the present invention provides a skin-regenerating composition containing the fibroblasts cultured according to said method.
  • FIG. 1 is a graphic diagram showing the proliferation rate of fibroblasts cultured according to Example 1.
  • FIG. 2 is a graphic diagram showing the results of quantitative analysis of collagen according to Example 1.
  • FIG. 3 is a graphic diagram showing the results of quantitative analysis of glycosamonoglycan (GAG) according to Example 1.
  • FIG. 4 is a photograph showing the results of analysis of COLlAl mRNA and
  • FIG. 5 is a graphic diagram showing the proliferation rate of fibroblasts cultured according to Example 2.
  • FIG. 6 is a graphic diagram showing the results of quantitative analysis of collagen according to Example 2.
  • FIG. 7 is a graphic diagram showing the results of quantitative analysis of glycosamonoglycan (GAG) according to Example 2.
  • FIG. 8 is a photograph showing the results of analysis of COLlAl mRNA and GADPH mRNA according to Example 2.
  • FIG. 9 is a photograph showing a nude mouse in which a fibroblast filler according to Example 3 was formed.
  • FIG. 10 is a photograph showing the fibroblast filler in the nude mouse of FIG.
  • FIG. 11 is a photomicrograph showing a filler of control group B in Example 3.
  • FIG. 12 is a photomicrograph showing a filler of experimental group Bl in Example 3.
  • FIG. 13 is a photomicrograph showing a filler of experimental group B2 in
  • FIG. 14 is a photomicrograph showing a filler of experimental group B3 in Example 3.
  • FIG. 15 is a graphic diagram showing the results of quantitative analysis of collagen in the filler according to Example 3.
  • FIG. 16 is a graphic diagram showing the results of quantitative analysis of glycosamonoglycan (GAG) in the filler according to Example 3.
  • FIG. 17 is a photograph showing the results of analysis of COLlAl mRNA and GADPH mRNA in the filler according to Example 3.
  • a method for culturing fibroblasts according to the present invention comprises the steps of: (A) preparing fibroblasts; (B) culturing the fibroblasts; (C) incubating the fibroblasts; and (D) isolating the fibroblasts.
  • a method for culturing fibroblasts according to the present invention comprises the steps of: (A) preparing fibroblasts; (B) culturing the fibroblasts; (C) incubating the fibroblasts; and (D) isolating the fibroblasts.
  • Fibroblasts are collagen-producing cells, which undergo cell division to increase the number thereof.
  • the fibroblasts can be prepared using a conventional isolation method, which can be used in the art.
  • Preferred are autologous dermal fibroblasts prepared from the skin tissue from subjects (patients) themselves. Such autologous dermal fibroblasts do not cause rejection and have a very excellent histocompatibility.
  • the skin tissue isolated from the subjects is washed several times with a substance containing an antibacterial or antifungal agent. This aims to prevent contamination from occurring in a subsequent step of culturing the fibroblasts.
  • Substances used in the washing process may include media such as DMEM (Dulbecco's Modified Eagle's Medium) and IMDM (Iscove's Modified Dulbecco's Medium).
  • the fibroblasts can be treated with EDTA- trypsin as known in the art, or can be processed for frozen storage.
  • the culture medium a conventional medium such as DMEM (Dulbecco's Modified Eagle's Medium) or IMDM (Iscove's Modified Dulbecco's Medium) can be used.
  • the medium may contain or not contain autologous serum.
  • the medium further contains a placenta extract.
  • the placenta has activities such as oxygen supply, poison neutralization and hormone secretion in the mother's body, and the active ingredients thereof include amino acids, vitamins, minerals, a number of enzymes and nucleic acids.
  • the placenta is not limited to that of specific mammals such as human beings or cattle, and any mammalian placenta can be suitably used in the present invention.
  • An extract from the placenta inhibits wrinkle formation, promotes collagen production, and also is effective for the proliferation of a large amount of fibroblasts.
  • placenta extract one prepared according to a conventional method known in the art is used. It is preferable to use a placenta extract commercially available from Melsmon Pharmaceutical Co., Ltd.
  • concentration of the placenta extract used in the culture of the fibroblasts is preferably 0.05-20%. It is more preferable to use both the placenta extract and bovine fetal serum.
  • high-frequency waves can also be used to promote the proliferation of the cells.
  • (C) Step of incubating fibroblasts The cultured fibroblasts are treated with trypsin and transferred into a new flask, in which the fibroblasts are subcultured.
  • the flask can also be split at a ratio of 1 : 3 or 1 : 5.
  • Preferred is a triple bottom T-150 flask having a total culture area of 450 cm 2 for expanding fibroblsts.
  • the flask has a capacity to yield about 6 x 10 6 to 1.8 x 10 7 cells.
  • the growth medium is replaced with a serum-free medium.
  • the cells are typically incubated at a temperature of 30-40 ° C for 10-20 hours.
  • the serum-free medium When the cells are incubated in the serum-free medium, proteins derived from non-autologous serum added to the medium can be removed.
  • DMEM medium with about 2 mM glutamine, with or without about 110 mg/L sodium pyruvate, can be used.
  • the serum- free medium also can contain one or more antibiotics.
  • the fibroblasts are isolated from the medium using trypsin-EDTA.
  • the isolated fibroblasts are centrifuged or resuspended, and sufficiently washed.
  • the washed fibroblasts are mixed with the same amount of a saline solution to form a skin- regenerating composition.
  • the composition may further comprise fibrin and a pharmaceutically acceptable carrier.
  • the inventive composition containing the fibroblasts cultured using the placenta extract is injected for the repair and regeneration of subcutaneous or dermal tissues, such as wrinkles, abdominal stria, depressed scars, and nontraumatic depressions of the skin. Also, it can be widely used for other plastic purposes.
  • Example 1 Culture of fibroblasts using placenta extract Fibroblasts of control and experimental groups were cultured and isolated in conditions shown in Table 1 below. Table 1
  • Dermis was isolated from the skin below the ear of 20-year-old healthy men.
  • the skin collected in the step (A) was washed several times with PBS (phospahate buffer saline, pH 7.4), and completely dissolved in 0.2% Type I collagenase at 4 ° C overnight.
  • the solution was centrifuged at 1000 rpm for 5 minutes to precipitate cells, and the cells were suspended in DMEM (Dulbecco's modified Eagle's medium; Gibco/BRL, USA) containing 100 units/mL penicillin G, 100 ⁇ g/mL streptomycin sulfate and 10% fetal bovine serum (Gibco/BRL, USA). Then, the medium was placed into a 100 mm culture dish, in which it was primarily cultured in 5% CO 2 at 37 "C .
  • DMEM Dulbecco's modified Eagle's medium
  • Gibco/BRL Gibco/BRL, USA
  • the culture medium was treated with a trypsin-EDTA solution to isolate the fibroblasts. Then, the fibroblasts were seeded into a 6-well plate at a concentration of 1 x 10 5 /ml and maintained in serum starvation conditions for at least 12 hours. Then, a placenta extract (Melsmon Pharmaceutical Co., Ltd., Japan) was added to the fibroblasts of experimental groups Al -A3 except for the fibroblasts of control group A, at concentrations of 0.1%, 0.5% and 1%, respectively, followed by culture.
  • a placenta extract (Melsmon Pharmaceutical Co., Ltd., Japan) was added to the fibroblasts of experimental groups Al -A3 except for the fibroblasts of control group A, at concentrations of 0.1%, 0.5% and 1%, respectively, followed by culture.
  • the cultured fibroblasts were treated with trypsin and transferred into a new triple bottom flask in which they were subcultured.
  • the previous growth medium was replaced with a serum-free medium, in which the fibroblasts were incubated at 35 ° C for 20 hours.
  • the fibroblasts were isolated from the medium using trypsin-EDTA. The isolated fibroblasts were centrifuged or resuspended, and sufficiently washed. The fibroblasts of control group A and experimental groups Al -A3 thus prepared were analyzed in the following manner.
  • GAG glycosaminoglycan
  • collagen which are cellular matrixes secreted by fibroblasts
  • DMMB 1.9 dimethylmethylene blue chloride
  • RNA isolation kit Fluka, USA
  • reverse transcriptase-polymerase chain reaction PCR
  • PCR polymerase chain reaction
  • the genes amplified by RT-PCR in this experiment were a glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) gene and a collagen type I gene (COLlAl).
  • GAPDH glyceraldehyde-3 -phosphate dehydrogenase
  • COLlAl collagen type I gene
  • the GAPDH gene was used as an index indicative of the suitability of PCR, because it was expressed in all the samples.
  • 5'-GGGCTGCTTTTAACTCTGGT-S' was used as a forward primer
  • 5'-TGGCAGGTTTTTCTAGACGG-S' was used as a backward primer.
  • the PCR amplification was performed in the following conditions: 35 cycles each consisting of 30 sec of at 94 ° C, 30 sec at 60 ° C and 30 sec at 72 °C ; and then a final extension of 7 min at 72 ° C .
  • 5'-CTCCGGCTCCTGCTCCTCTTA-S' was used as a forward primer
  • 5 '-GCACAGCACTCGCCCTCCC-3 ' was used as a backward primer.
  • the PCR amplification was performed in the following conditions: 35 cycles each consisting of 30 sec at 94 °C, 30 sec at 56 0 C and 1 min at 72 ° C ; and then a final extension of 7 min at 72 0 C .
  • the products resulting from the RT-PCR reactions were separated through 1.5% agarose gel electrophoresis, were stained with ethidium bromide, and were then photographed under UV light to observe the expression of each of the genes.
  • the analysis results for each of the genes are as follows.
  • the expression of the COLlAl gene could be observed in all the control group A and the experimental groups Al -A3.
  • the expression level of the COLlAl gene was the lowest in the control group A and was higher in the order of the experimental groups Al, A2 and A3.
  • Example 2 Culture of fibroblasts using both placenta extract and bovine fetal serum Fibroblasts of control group B and experimental groups B1-B3 were cultured and isolated in conditions shown in Table 2 below. The proliferation and biological index of the isolated fibroblasts were examined in the same manner as in Example 1. Table 2
  • the proliferation rate of the fibroblasts was higher in the experimental groups B1-B3 than in the control group B.
  • the difference in proliferation rate between the experimental groups and the control group was statistically significant (p ⁇ 0.05).
  • the experimental group Bl showed a relatively low proliferation rate, but the difference between the experimental groups was not statistically significant (p>0.05).
  • the expression level of the biological index of the fibroblasts was higher in all the experimental groups B1-B3 than in the control group.
  • the amounts of extracellular matrix collagen and glycosamonoglycan (GAG) were the most highest in the experimental groups B2 and B3, and the increase in the matrixes of the experimental groups B1-B3 compared to the control group B was statistically significant (p ⁇ 0.05).
  • the expression of COLlAl could be observed in all the control group B and the experimental groups B1-B3.
  • the expression level of COLlAl was the lowest in the control group B, and the highest in the experimental groups B2 and B3.
  • the use of the placenta extract or the use of both the placenta extract and the bovine fetal serum had a great effect on fibroblast proliferation and gene expression.
  • Example 3 Construction of f ⁇ broblast-containing injection and formation of filler
  • the injected fibroblasts were visually observed and the tissue thereof was observed with a microscope. Also, the biological index thereof was analyzed.
  • the preparation, culture and incubation of the fibroblasts were carried out in the same manner as in Example 2.
  • the isolation of the fibroblasts and the construction of the fibroblast injection were carried out in the following manner.
  • Isolation of fibroblasts and construction of fibroblast injection A sufficient amount of fibroblasts were prepared, and the medium containing the fibroblasts was then treated with a trypsin-EDTA solution to isolate the fibroblasts from the culture dish and centrifuged at 1000 rpm for 5 minutes to precipitate the cells.
  • a fibroblast injection was constructed using fibrin glue gel as a cell support.
  • the cells were added to an injector containing a fibrinogen solution, one component of fibrin glue, at a concentration of 1 x 10 6 cells/ml, and the injector was placed in a crosslinking injection case which was constructed such that fibrinogen could be crosslinked when the injector was injected together with another injector containing a thrombin solution, another component fibrin glue, thereby constructing a fibroblast injection.
  • the constructed injection was injected into animals in the following manner.
  • mice 4-6-week-old nude mice were anesthetized with 0.3-0.5 mg of avertin per g of body weight, and then 1 ml of each of the fibroblast injections of the control group and the experimental groups Bl -B 3 was injected into the subcutaneous layer of both sides of the mouse back. 8 weeks after the injection, the animals were euthanized and a soft tissue filler of the injected fibroblasts was collected from each of the animals and analyzed in the following manner.
  • the volume and weight of each of the obtained fibroblast soft-tissue filler masses were measured, and the measurement results were compared between the control group and the experimental groups.
  • the mRNA expression patterns of the fillers were analyzed in the same manner as in Example 1.
  • FIG. 9 shows a nude mouse in which the fibroblast filler according to the experimental group B 3 was formed
  • FIG. 10 shows the fibroblast filler in the nude mouse of FIG. 9.
  • FIGS. 11 to 14 are photomicrographs of the experimental groups Bl, B2 and B3, respectively.
  • FIGS. 12 to 14 are photomicrographs of the experimental groups Bl, B2 and B3, respectively.
  • fibroblast extracellular matrix collagen was formed in an amount larger than the control group.
  • the expression of extracellular matrix collagen and glycosamonoglycan was higher in the order of the experimental groups B3, B2 and Bl.
  • the mRNA expression of collagen type I gene (COLlAl) was the highest in the experimental group B3 and lower in the order of the experimental groups Bl and B2, but the difference between the experimental groups B 1 and B2 insignificant

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Abstract

La présente invention se rapporte à un procédé de culture de fibroblastes, qui comprend les étapes consistant : (A) à préparer des fibroblastes ; (B) à cultiver des fibroblastes à l'aide d'un milieu de culture contenant un extrait placentaire ; (C) à incuber les fibroblastes cultivés dans un milieu exempt de sérum ; et (D) à isoler les fibroblastes incubés, les fibroblastes isolés ne renfermant pas d'autres cellules que des fibroblastes et ne contenant pas de protéines sériques du milieu.
PCT/KR2006/004880 2005-11-25 2006-11-20 Procede de culture de fibroblastes faisant appel a un extrait placentaire et composition de regeneration de la peau associee WO2007061205A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
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CN102791278A (zh) * 2010-02-26 2012-11-21 现代细胞与组织技术公司 包括胎盘提取物的细胞保护剂
WO2013166045A1 (fr) * 2012-04-30 2013-11-07 The Johns Hopkins University Procédés pour utiliser des fibroblastes autologues pour modifier l'identité cutanée
CN105969719A (zh) * 2016-07-19 2016-09-28 安徽惠恩生物科技股份有限公司 一种皮肤成纤维细胞提取制备方法
CN113215084A (zh) * 2021-06-11 2021-08-06 中国农业科学院兰州兽医研究所 一种羊胎儿皮肤成纤维细胞、其分离方法与应用

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WO2013166045A1 (fr) * 2012-04-30 2013-11-07 The Johns Hopkins University Procédés pour utiliser des fibroblastes autologues pour modifier l'identité cutanée
CN105969719A (zh) * 2016-07-19 2016-09-28 安徽惠恩生物科技股份有限公司 一种皮肤成纤维细胞提取制备方法
CN113215084A (zh) * 2021-06-11 2021-08-06 中国农业科学院兰州兽医研究所 一种羊胎儿皮肤成纤维细胞、其分离方法与应用

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