WO2008002064A1 - Soft tissue filler composition for injection and preparation method thereof - Google Patents

Soft tissue filler composition for injection and preparation method thereof Download PDF

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Publication number
WO2008002064A1
WO2008002064A1 PCT/KR2007/003100 KR2007003100W WO2008002064A1 WO 2008002064 A1 WO2008002064 A1 WO 2008002064A1 KR 2007003100 W KR2007003100 W KR 2007003100W WO 2008002064 A1 WO2008002064 A1 WO 2008002064A1
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Prior art keywords
dermal
cells
injection
autologous
filler composition
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PCT/KR2007/003100
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French (fr)
Inventor
Bin Han
Jay Do Choi
Min Jung Nam
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S-Biomedics Co., Ltd.
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Publication of WO2008002064A1 publication Critical patent/WO2008002064A1/en

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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
    • 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
    • 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
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides

Definitions

  • bovine collagen The preparation and application of bovine collagen have been disclosed in U.S. Patent Nos. 3,949,073, 4,424,208 and 4,488,911.
  • the bovine collagen came into the market under the trade name Zyderm I , II and HI, wherein the content of collagen is in the range from 35 mg/ml to 65 mg/ml.
  • Zyderm since an antibody to the bovine collagen is generated in 90% of the patients being treated with Zyderm (and 1 to 3% thereof shows clear allergic reactions), Zyderm does not provide satisfactory results.
  • injectable glutaraldehyde cross-linked collagen has been developed and sold under the trade name Zyplast. Said protein does not cause any allergic reactions, but its viscosity is too strong to obtain good therapeutic effects (U.S. Patent Nos. 4,582,640 and 4,642,117).
  • U.S. Patent Nos. 4,969,912 and 5,332,802 disclose a method of using autologous injectable human collagen for preventing an immune reaction due to the use of bovine collagen, which is available under the trade name AUTOLOGEN.
  • Fibrel is based on a porcine collagen. Three items are combined to actually make up Fibrel, i.e., porcine gelatin, aminocaproic acid and plasma from the subject being treated. Fibrel shows the desired effects in some patients. However, most of the patients being treated are subject to swelling at the injection site and it is impossible to keep up its therapeutic effects for a long time.
  • Artecoll is a complex of artificially synthesized particles and bovine collagen, which has been safely used in Canada, Mexico and Europe for several years. Artecoll exhibits prolonged therapeutic effects by providing autologous collagen through the stimulation of regional dermis. However, it has also problems in that the subject being treated can feel the presence of the particles and the swelling may occur at the treatment site.
  • the effective ingredient of the injection disclosed in Chinese Patent No. 03155833.X is obtained by culturing the autologous skin sample in a medium partly supplemented with animal-derived serum.
  • a medium partly supplemented with animal-derived serum there are several drawbacks to this method.
  • the content of collagen in said injection which is responsible for the immediate onset of therapeutic effects, is too small, the effects for removing wrinkles and scars are late in terms of exertion, thereby taking 4 to 6 months to obtain satisfactory therapeutic effects.
  • a subject being treated may worry about the safety due to the use of animal- derived serum as a component of the culture medium.
  • compositions disclosed in said patents essentially consist of autologous muscle cells, autologous UMC or autologous keratinocytes, which are entirely different from a soft tissue filler composition comprising dermal fibroblast stem cells, dermal fibroblast transit amplifying cells and dermal fibroblasts described below.
  • a method of preparing a soft tissue filler composition for injection which comprises the steps of:
  • the autologous dermal biopsy used in Step 1) is prepared by disinfecting a target site on the skin of the patient being treated with alcohol, anesthetizing topically, excising epidermis and dermis in a size of 1 to 30 mm 2 therefrom, and then storing the same in a tissue stock solution.
  • the skin tissue useful for the in vitro serum-free cultivation and proliferation of autologous dermal cells in this step may include all the skin, epidermis and dermis obtained from the back of the ear, eyebrows, the lower part of the eyes and other regions.
  • prepared autologous dermal tissue sample is subjected to tissue digestion and cell isolation procedures.
  • the basal medium useful for the present invention may be prepared by using conventional medium constitutions well known to one of ordinary skill in the art, e.g., ⁇ Cell Experimental Protocols>(2001, Science Press, Chief editor : D.L. Speycutter, Translation: Huang Peitang).
  • the dermal cells isolated from the autologous dermal tissue are inoculated into the serum-free culture medium and cultured in an incubator at 37 °C under 5% CO 2 for 6 to 10 weeks. At this time, it is preferable to replace the medium with a fresh one at 3 day intervals.
  • the autologous dermis-derived cell culture solution prepared above is centrifuged at a temperature ranging from 4 to 32 ° C at a speed ranging from 800 to 1,200 rpm/min to remove a supernatant, thereby recovering only an autologous dermis-derived cell pellet containing proliferated dermal fibroblast stem cells, dermal fibroblast transit amplifying cells, dermal fibroblasts and collagen secreted therefrom.
  • the cultured and proliferated dermal fibroblast stem cells, dermal fibroblast transit amplifying cells and dermal fibroblasts and collagen secreted therefrom according to the present invention may be confirmed as follows:
  • Alexa 594 Anti-Rabbit IgG Antibody, Molecular probe
  • Alexa 488 Anti-Mouse IgG Antibody, Molecular probe
  • the washed slide glass was observed with a fluorescent microscope (Olympus
  • the dermis-derived cells cultured according to the method of the present invention exhibit a different expression pattern of cell markers from the adipose tissue-derived cells and display nestin (a marker for neural progenitor cells), they show immunological characteristics corresponding to neural progenitor-derived stem cells and not mesenchymal-derived stem cells (Toma et al., Stem Cells 23: 727-737, 2005; Fernandes et al., Nature 6: 1082-1093, 2004; and Toma et al., Nature 3: 778- 784, 2001).
  • DAPI 4,',6-Diamidin-2-phenylindole dihydrochloride, SIGMA
  • PBS fetal bovine serum
  • DAPI 4,6-Diamidin-2-phenylindole dihydrochloride
  • the slide glass was then soaked in the DAPI solution to stain the cells and washed three times with DPBS for 10 minutes.
  • the washed slide glass was observed with a fluorescent microscope (Olympus IX 71) and recorded with a digital camera system.
  • nestin and vimentin were expressed in the dermis-derived cells of the present invention.
  • the cells expressing both of them were observed.
  • the expression of vimentin was detected, but nestin was not expressed.
  • Alexa 594 Anti-Rabbit IgG Antibody, Molecular probe
  • Alexa 488 Anti-Mouse IgG Antibody, Molecular probe
  • the dermis-derived cell culture material obtained by culturing in a serum-free medium according to the present invention shows signals for fibronectin and FSP-I known as a fibroblast marker protein as well as for nestin known as a neural progenitor-derived stem cell marker protein, which are clearly different from mesenchymal-derived stem cells such as adipose tissue-derived cells.
  • the dermis-derived cells were cultured in a serum-free culture medium for 2 weeks according to the same method as described in Example 5.
  • 0.25% trypsin/EDTA solution was added to the culture solution to detach the cells from the culture plate, followed by RNA extraction by using a RNA extraction kit (Purelink Micro to-midi, Invitrogen).
  • a reaction solution was prepared by mixing thus extracted RNA, dNTP and oligo-dT 20 mer primer and adjusting its final volume to 10 ⁇ Jt.
  • the reaction solution was incubated at 65 ° C for 5 minutes, followed by keeping at 0 ° C for a minute.
  • 10 ⁇ i of a cDNA synthetic mixture (1OxRT buffer, 25 mM MgCl 2 , 0.1 M DTT, RNase OUT, Superscript IH RT) was added thereto, the resulting solution was subjected to reverse transcription at 50 ° C for 50 minutes and 85 ° C for 20 minutes.
  • To the reaction solution was added 1 ⁇ Jt of RNAse H and kept at 37 ° C for 20 minutes to thereby synthesize cDNA.
  • SEQ ID NO: 7 of pax3-l and SEQ ID NO: 8 of ⁇ ax3-2 for Pax3 amplification SEQ ID NO: 9 of snail- 1 and SEQ ID NO: 10 of snail-2 for Snail amplification
  • the soft tissue filler composition for injection of the present invention exerts prolonged therapeutic effects without causing any immune response and side-effect due to the use of autologous dermal cells. Further, since it completely rules out the risk of the use of animal-derived serum through in vitro serum-free cultivation and immediately shows therapeutic effects by producing a large quantity of collagen, the soft tissue filler composition for injection of the present invention can be effectively used for improving the skin tone and resilience as well as smoothing and removing wrinkles and scars.

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Abstract

The present invention relates to a method of preparing a soft tissue filler composition for injection, which comprises the steps of: 1) digesting an autologous dermal tissue isolated from the patient's own skin and separating into single cells; 2) culturing and proliferating the isolated dermal cells via in vitro serum-free cultivation to obtain an autologous dermis-derived cell culture material containing dermal fibroblast stem cells, dermal fibroblast transit amplifying cells, dermal fibroblasts and collagen; 3) centrifuging the autologous dermis-derived cell culture material to separate an autologous dermis-derived cell pellet; and 4) suspending the autologous dermis-derived cell pellet in a glucose injection or a random injection to obtain a suspension for injection; and a soft tissue filler composition for injection prepared by the same.

Description

SOFT TISSUE FILLER COMPOSITION FOR INJECTION AND PREPARATION METHOD THEREOF
Field of the Invention
The present invention relates to a soft tissue filler composition for injection, which is prepared by digesting an autologous dermal tissue isolated from the patient's own skin, separating into single cells, in vitro culturing and proliferating the separated cells in a serum-free medium. The present invention further relates to a method of preparing the above composition.
Background of the Invention
Typical filling methods used for repairing skin defects are divided into two groups, i.e., dermal filling and subcutaneous filling. All filler components used in said methods generally contain biological synthetic materials or heterologous proteins than being entirely composed of autologous tissue-derived activators. Paraffin was used for the first time as a filling material in the 19th century. However, it gave rise to too many side effects and its filling effect was not satisfactory. Thereafter, a method of employing exogenous proteins as a filling material was developed. In particular, it was found that a filling agent using bovine collagen exhibits a rapid onset of therapeutic effect. However, there was a problem in that once implanted, the collagen is gradually absorbed back into the skin after 3 to 6 months. The preparation and application of bovine collagen have been disclosed in U.S. Patent Nos. 3,949,073, 4,424,208 and 4,488,911. The bovine collagen came into the market under the trade name Zyderm I , II and HI, wherein the content of collagen is in the range from 35 mg/ml to 65 mg/ml. However, it has been found that since an antibody to the bovine collagen is generated in 90% of the patients being treated with Zyderm (and 1 to 3% thereof shows clear allergic reactions), Zyderm does not provide satisfactory results. In order to solve these problems, injectable glutaraldehyde cross-linked collagen has been developed and sold under the trade name Zyplast. Said protein does not cause any allergic reactions, but its viscosity is too strong to obtain good therapeutic effects (U.S. Patent Nos. 4,582,640 and 4,642,117).
Further, U.S. Patent Nos. 4,969,912 and 5,332,802 disclose a method of using autologous injectable human collagen for preventing an immune reaction due to the use of bovine collagen, which is available under the trade name AUTOLOGEN. However, its therapeutic effect is maintained only for a few months. Fibrel is based on a porcine collagen. Three items are combined to actually make up Fibrel, i.e., porcine gelatin, aminocaproic acid and plasma from the subject being treated. Fibrel shows the desired effects in some patients. However, most of the patients being treated are subject to swelling at the injection site and it is impossible to keep up its therapeutic effects for a long time. BOTOX, which was approved by FDA on April, 2002 to undergo clinical trials, is an injection solution for temporarily removing wrinkles. BOTOX shows therapeutic effects on the removal of wrinkles by anesthetizing facial muscle and has several advantages such as easy manipulation and immediate onset of therapeutic effects. However, there are numerous problems in that the patient's facial expression becomes hardened after the treatment, the duration of therapeutic effect is too short. Further, there is a risk of facial paralysis.
Artecoll is a complex of artificially synthesized particles and bovine collagen, which has been safely used in Canada, Mexico and Europe for several years. Artecoll exhibits prolonged therapeutic effects by providing autologous collagen through the stimulation of regional dermis. However, it has also problems in that the subject being treated can feel the presence of the particles and the swelling may occur at the treatment site.
As mentioned above, said heterologous filling materials cause side effects such as the possibility of allergic and rejection reactions and the limited duration of therapeutic effects.
U.S. Patent Nos. 5,591,444, 5,665,372 and 5,660,850 assigned to Isolagen Technologies, Inc. describe a method of treating defects of skin and soft tissue by using autologous dermal fϊbrocytes. In particular, said patents disclose a method of preparing an injection by: providing autologous skin tissue from a subject being treated; in vitro culturing the same in a bovine serum containing medium; and packaging the cultured autologous fibrocytes in a Ringer's solution. Said injection does not cause any allergic and heterologous rejection reactions, but it is not free from the safety problem due to the use of animal-derived serum. Further, since the Ringer's solution is not favorable to the prolonged activity maintenance of an effective ingredient, said injection must be used within a short timeframe after the preparation thereof, which causes inconvenience in terms of actual use.
Beijing Yiling Bioengineering Co., Ltd has developed a method of treating wrinkles and scars by using an injection, which is prepared by: isolating autologous skin from a subject being treated; in vitro culturing and proliferating the same; and suspending the cultured cells in a glucose injection solution (Ringer's solution) or a solution corresponding thereto (Chinese Patent No. 03155833.X).
The effective ingredient of the injection disclosed in Chinese Patent No. 03155833.X is obtained by culturing the autologous skin sample in a medium partly supplemented with animal-derived serum. However, there are several drawbacks to this method. First, since the content of collagen in said injection, which is responsible for the immediate onset of therapeutic effects, is too small, the effects for removing wrinkles and scars are late in terms of exertion, thereby taking 4 to 6 months to obtain satisfactory therapeutic effects. Further, a subject being treated may worry about the safety due to the use of animal- derived serum as a component of the culture medium.
Further, International Publication No. WO 2003/094837 discloses a composition containing autologous, passaged fibroblasts and muscle cells (optionally, biodegradable acellular matrix components/biodegradable acellular fillers (e.g., collagen)). It further discloses a method of repairing tissues, which have been degenerated in a subject as a result of a disease, disorder or defect. Further, International Publication No. WO 2004/048557 describes a composition, comprising: (i) autologous undifferentiated mesenchymal cells (UMC) and autologous fibroblasts; (ii) autologous UMC; or (iii) autologous fibroblasts and autologous keratinocytes as an effective ingredient. It also discloses a method for the preparation thereof and a method for repairing tissues by using the same. However, all the compositions disclosed in said patents essentially consist of autologous muscle cells, autologous UMC or autologous keratinocytes, which are entirely different from a soft tissue filler composition comprising dermal fibroblast stem cells, dermal fibroblast transit amplifying cells and dermal fibroblasts described below.
Therefore, the present inventors have endeavored to overcome the prior art problems and developed an autologous dermis-derived cell culture material containing dermal fibroblast stem cells, dermal fibroblast transit amplifying cells and dermal fibroblasts obtained through in vitro serum-free cultivation from autologous skin tissue together with an abundance of collagen as a complex filler. Further, it has been confirmed that thus prepared complex filler exhibits therapeutic effects of removing and recovering wrinkles and scars within a short timeframe and maintains these therapeutic effects for a long time.
Disclosure
Technical Problem
Accordingly, the primary object of the present invention is to provide a novel soft tissue filler composition for injection in which there is no problem in terms of safety because it does not use animal-derived serum. It also seeks to provide a composition, which is very useful for removing wrinkles and scars through the immediate onset and prolonged maintenance of therapeutic effects, and which can be effectively used for the improvement of skin tone and resilience. The present invention is also directed to a method of preparing the above composition.
Technical Solution
In accordance with one aspect of the present invention, there is provided a method of preparing a soft tissue filler composition for injection, which comprises the steps of:
1) digesting an autologous dermal biopsy isolated from the patient's own skin and separating into single cells;
2) culturing and proliferating the isolated dermal cells via in vitro serum-free cultivation to obtain an autologous dermis-derived cell culture material containing dermal fibroblast stem cells, dermal fibroblast transit amplifying cells, dermal fibroblasts and collagen;
3) centrifuging the autologous dermis-derived cell culture material to separate an autologous dermis-derived cell pellet; and 4) suspending the autologous dermis-derived cell pellet in a glucose injection or a random injection to obtain a suspension for injection. In accordance with another aspect of the present invention, there is provided a soft tissue filler composition for injection prepared by said method.
Hereinafter, the present invention is described in more detail.
First, the autologous dermal biopsy used in Step 1) is prepared by disinfecting a target site on the skin of the patient being treated with alcohol, anesthetizing topically, excising epidermis and dermis in a size of 1 to 30 mm2 therefrom, and then storing the same in a tissue stock solution. The skin tissue useful for the in vitro serum-free cultivation and proliferation of autologous dermal cells in this step may include all the skin, epidermis and dermis obtained from the back of the ear, eyebrows, the lower part of the eyes and other regions. Thus prepared autologous dermal tissue sample is subjected to tissue digestion and cell isolation procedures. In particular, the autologous dermal tissue sample may be placed in a culture dish and subjected to tissue digestion by treating with a pancreatin/EDTA solution. Thereafter, the tissue digestion may be stopped by adding a DMEM solution containing 10% FBS. The digested tissue in the reaction solution may be broken and isolated into single cells by applying gentle suction repeatedly using a suction pipe and centrifuged to remove a supernatant to thereby obtain a cell pellet. In Step 2), the cells prepared in Step 1) are cultured in vitro in a serum- free medium according to a conventional method in the art such as a tissue digestion culture method or a tissue fragment culture method.
The cell culture method of the present invention is different from the prior art methods in terms of using a serum-free culture medium. In particular, the cell culture method of the present invention is characterized by: using a serum-free culture medium supplemented with growth factors (e.g., epidermal growth factor and dermal growth factor) and activation factors (e.g., cortical hormone and bovine pituitary extract) instead of adding animal-derived serum such as bovine serum to a basal medium; culturing cells in the serum-free culture medium for 6 to 10 weeks; and inducing cell proliferation and sufficient secretion of collagen. It has been confirmed that the activity and morphology of the cells cultured in the serum-free culture medium containing growth factors and activation factors in place of animal-derived serum according to the method of the present invention are normal. Especially, thus obtained culture solution contains a large quantity of collagen as well as the autologous dermis-derived cell culture material containing dermal fibroblast stem cells, dermal fibroblast transit amplifying cells and dermal fibroblasts as an effective ingredient. In case of preparing a filler using the culture solution, the content of collagen in the filler may range from 10 to 100 mg/m£. The basal medium useful for the present invention may be prepared by using conventional medium constitutions well known to one of ordinary skill in the art, e.g., <Cell Experimental Protocols>(2001, Science Press, Chief editor : D.L. Speycutter, Translation: Huang Peitang).
The serum-free culture medium, growth factors and activation factors employable in the present invention may include all products commercially available in the art. The growth factors, which can be added to the serum-free culture medium of the present invention, may include epidermal growth factor (EGF), dermal growth factor, basic fibroblast growth factor (bFGF) and the like. Said factors can be used alone or in the form of a mixture thereof. The concentration of the growth factors in the serum-free culture medium may preferably range from 0.1 to 5 ng/m#, and more preferably from 0.5 to 2 ng/ml. Further, the activation factors, which can be added to the serum-free culture medium of the present invention, may include cortical hormone, bovine pituitary extract (BPE), insulin, hydrocortisone and the like. Said factors can be used alone or in the form of a mixture thereof. The concentration of the activation factors in the serum-free culture medium may preferably range from 0.1 to 1 %, and more preferably from 0.2 to 0.5%.
The dermal cells isolated from the autologous dermal tissue are inoculated into the serum-free culture medium and cultured in an incubator at 37 °C under 5% CO2 for 6 to 10 weeks. At this time, it is preferable to replace the medium with a fresh one at 3 day intervals. In Step 3), the autologous dermis-derived cell culture solution prepared above is centrifuged at a temperature ranging from 4 to 32 °C at a speed ranging from 800 to 1,200 rpm/min to remove a supernatant, thereby recovering only an autologous dermis-derived cell pellet containing proliferated dermal fibroblast stem cells, dermal fibroblast transit amplifying cells, dermal fibroblasts and collagen secreted therefrom.
The cultured and proliferated dermal fibroblast stem cells, dermal fibroblast transit amplifying cells and dermal fibroblasts and collagen secreted therefrom according to the present invention may be confirmed as follows:
Dermal fibroblast stem cells: They can be analyzed according to the BrDU (5-Bromo-Deoxy-Uridine) antibody method described in the literature
(Huang hui (ft#ϊ) , Lai Xinan ($M l^) , Wang Zhengguo (ΞE _E HI) f
Wang LiIi (ΞEBβ ββ) ? Roles of a material during the wound healing in transferring to dermal stem cells and delivering a receptor; Chinese Journal of
Trauma, 2004, 20(3): 142-145). The standard morphology of the dermal fibroblast stem cells observed with an optical microscope under 10Ox magnifications is a thin, long roll-shape. They are stained to dark brown according to the BrdU antibody method (see Fig. 1).
Dermal fibroblast transit amplifying cells: The standard morphology of the dermal fibroblast transit amplifying cells observed with an optical microscope under 10Ox magnifications is a thin and small, long roll-shape similar to the dermal fibroblast stem cells. They are stained to brown according to the BrdU antibody method (see Fig. 2).
Dermal fibroblasts: The standard morphology of the dermal fibroblasts observed with an optical microscope under 100χ magnifications is a long roll- shape having many processes (see Fig. 3).
Collagen: The concentration of collagen may be measured according to the collagen measurement method described in the literature (Ministry of Health P.R.China, Pharmacopoeia Committee, <Chinese Pharmacopoeia> (2nd Ed.), Beijing Chemical Industry Press, 2000). Step 4) is to prepare a soft tissue filler composition for injection by suspending the autologous dermis-derived cell pellet comprising the effective ingredients separated above in a glucose injection at a concentration of 5% or a common injection. At this time, thus prepared suspension may further include suitable additive ingredients. The soft tissue filler composition of the present invention comprises the cultured dermal fibroblast stem cells, dermal fibroblast transit amplifying cells and dermal fibroblasts isolated from the patient's own tissue together with collagen secreted there from as an effective ingredient. The final concentration of the effective cells in the injection preferably ranges from 1 x 107 to 8x 107 cells/m-C, and the content of collagen preferably ranges from 10 to 100 mg/m£, more preferably 20 to 60 mg/m-C.
The soft tissue filler composition of the present invention may be formulated into injectable products intended for human skin, which can be accomplished according to a conventional method well known to one of ordinary skill in the art. For example, the composition of the present invention may be in the form of a solution, thick solution, suspension or gel. Said composition may further comprise suitable excipients adapted for injection into skin. Suitable excipients should be well tolerated, stable and yield a consistency that allows for easy and pleasant utilization. Here, suitable examples of an excipient include, but are not limited to, phosphate buffered saline, bacteriostatic saline, propylene glycol, starch, sucrose and sorbitol.
Further, the soft tissue filter composition of the present invention may further comprise an additional agent, such as an inert and pharmaceutically acceptable carrier or diluent, an excipient, a thickening agent, an emulsifying agent, a preservative and a mixture thereof. Suitable examples of the above additional agents typically include those agents commonly used in pharmaceutical and skin care preparations. More specifically, such examples of an inert and pharmaceutically acceptable carrier or diluent include, but are not limited to, saline and purified water. Suitable thickening agents include acrylamides copolymer, carbomer, hydroxyethylcellulose, hydroxypropylcellulose, polyacrylic acid, polymethacrylic acid and polyvinyl alcohol, but are certainly not limited thereto. Suitable emulsifying agents include caprylic/capric triglyceride, ceteareth-7, cetyl alcohol, cetyl phosphate, isostearate-11 and sodium isostearate, but are not limited thereto. Preservatives impart to the composition of the present invention resistance to microbial attack and toxicity to microbes. Suitable examples include alcohols, any of the parabens, diazolidinyl urea, DMDM hydantoin, phenoxyethanol, and iodopropyryl butylcarbamate, but are not limited thereto. Examples of the above additional agents, other than those that are listed, may also be used in embodiments of this invention, as would be well appreciated by one of ordinary skill in the art.
The soft tissue filler composition for injection prepared according to the method of the present invention contains a large quantity of collagen, which functions to remove wrinkles and scars immediately at the injection area. Further, the other effective ingredients in the soft tissue filler composition for injection of the present invention play an important role in removing wrinkles and scars simultaneously with functioning to improve the local skin environment through the continuous production of collagen. This imparts youth and beauty to the subject being treated.
Accordingly, the soft tissue filler composition for injection of the present invention can be effectively used for not only recovering all types of wrinkles and scars on the face and neck, stretch marks from pregnancy, wrinkles on the back of the hand and the like, but also enhances the thickness, resilience and texture of the skin.
Since the soft tissue filler composition for injection of the present invention comprises the autologous dermis-derived cells as an effective ingredient, it does not cause any allergic and heterologous rejection reaction. Further, since the present invention does not use animal-derived serum during the preparation of the soft tissue filler composition for injection, there is no possibility of causing infectious diseases or side-effects. Accordingly, the soft tissue filler composition for injection of the present invention can immediately exhibit excellent cosmetic-therapeutic effects, endow natural facial expression to the subject being treated after the injection and maintain the expressed therapeutic effects for a long time.
Advantageous Effect
The soft tissue filler composition for injection of the present invention exerts prolonged therapeutic effects without causing any immune response and side-effect due to the use of autologous dermal cells. Further, since it completely rules out the risk of the use of animal-derived serum through in vitro serum-free cultivation and immediately shows therapeutic effects by producing a large quantity of collagen, the soft tissue filler composition for injection of the present invention can be effectively used for improving skin tone and resilience as well as smoothing and removing wrinkles and scars.
Description Drawings
The above and other objects and features of the present invention will become apparent from the following description of the invention taken in conjunction with the following accompanying drawings; which respectively show:
Fig. 1 is a photograph (x lOO) observed under an optical microscope showing dermal fibroblast stem cells proliferated by in vitro serum-free cultivation from autologous dermal cells according to the method of the present invention;
Fig. 2 is a photograph (x lOO) observed under an optical microscope showing dermal fibroblast transit amplifying cells proliferated by in vitro serum-free cultivation from autologous dermal cells according to the method of the present invention;
Fig. 3 is a photograph (x lOO) observed under an optical microscope showing dermal fibroblasts proliferated by in vitro serum-free cultivation from autologous dermal cells according to the method of the present invention; Fig. 4 is the result of comparing the morphology of autologous dermis- derived cell culture material (B) obtained according to the method of the present invention with that of adipose tissue-derived cells (A) by using a phase-contrast microscope;
Fig. 5 is the result of immunofluorescence assay comparing the expression patterns of nestin and fibronectin in autologous dermis-derived cell culture material (A) obtained according to the method of the present invention with those in adipose tissue-derived cells (B), wherein nestin is detected as a red spot at 594 nm, fibronectin is detected as a green spot at 488 nm and cell nuclei is detected as a blue spot by DAPI staining;
Fig. 6 is the result of immunofluorescence assay comparing the expression patterns of nestin and vimentin in autologous dermis-derived cell culture material (A) obtained according to the method of the present invention with those in adipose tissue-derived cells (B), wherein nestin is detected as a red spot at 594 nm, vimentin is detected as a green spot at 488 nm and cell nuclei is detected as a blue spot by DAPI staining; Fig. 7 is the result of immunofluorescence assay comparing the expression patterns of nestin and FSPl in autologous dermis-derived cell culture material (A) obtained according to the method of the present invention with those in adipose tissue-derived cells (B), wherein nestin is detected as a red spot at 594 nm, FSPl is detected as a green spot at 488 nm and cell nuclei is detected as a blue spot by DAPI staining; and
Fig. 8 is the result of reverse transcription-polymerase chain reaction (RT-PCR) analyzing the expression pattern of neural progenitor marker proteins in autologous dermis-derived cell culture material obtained according to the method of the present invention.
Best Mode
The present invention will now be described in more detail with reference to the following examples, which are not intended to limit the scope of the present invention.
<Example 1> Preparation of a soft tissue filler composition <1-1> Preparation procedure
After the back of the ear was disinfected with 70% alcohol and the local surface area thereon was anesthetized with 10% lidocaine and adrenalin
(1 : 100,000), the epidermal and dermal tissue sample was excised in a size of 4 mπf therefrom and stored in a stock solution (DMEM cell stock solution, Hyclone, USA).
The tissue sample was placed in a 35 mm culture dish, 2 ml of 0.05% pancreatin/EDTA solution was added thereto, and then the culture dish was kept in a CO2 incubator at 37 °C for 10 minutes to induce cell digestion. The digestion reaction was completed by adding 5 ml of DMEM supplemented with 10% FBS.
The digested tissue sample was cut into pieces and broken by gentle suction using a suction pipe to thereby separate into single cells. The separated dermal cells were centrifuged at 25 °C , 1000 φm/min for 5 minutes to remove a supernatant, thereby recovering only a cell pellet.
To the cell pellet obtained above was added 1 ml of a dermal cell culture medium (Cascade, Cat. No. 106, supplemented with 1 ng/ml of EGF as a growth factor and 0.2% of BPE as an activation factor, USA) and the mixture was subjected to primary culture in a CO2 incubator at 37 °C under 5% CO2.
The culture medium was replaced with a fresh one at intervals of 2 to 3 days and the primary cultured cells were subjected to secondary subculture until their confluence reaches the range from 50 to 70%. The resulting cell culture solution was digested with pancreatin, centrifuged to remove a supernatant and further cultured at a proliferative index of 1 :3
The culture solution obtained by the in vitro serum- free subculture for 4 to 6 weeks was centrifuged to separate a dermis-derived cell pellet, followed by suspending in a 5% glucose injection, thereby preparing 1 to 4 ml of a soft tissue filler composition for injection comprising the autologous dermis-derived cells at a concentration of 2* 107 to 6χ 107 cells/in^ as an effective ingredient.
<l-2> Tests for viral infection and immune response
To examine the presence of viral infection in the dermis-derived cell culture material obtained by culturing the autologous tissue sample for 4 to 6 weeks in Example <1-1>, 1 ml of the dermis-derived cell culture material was subjected to tests for HIV and hepatitis viral infection according to a KFDA standard protocol, which confirms that the cell culture material of the present invention is virus-free.
Further, it has been confirmed that the dermis-derived cell culture material does not occur any immune response through a subcutaneous experiment intended for the skin of the patient being treated by using 0.1 ml of the cell culture material. Here, such a subcutaneous experiment was conducted by modifying a penicillin allergy skin test, which decides a sample having no large area, red swelling phenomenon as a negative after applying 0.05 mi of a penicillin suspension to the epidermis on the inside of the wrist and observing for 30 minutes. In addition, as a result of aseptic test according to Chinese Biological Product Regulation (2000 Ed.), General Principle <Biological Product Aseptic Test Regulation^ Article A/B, it has been confirmed that the autologous dermis-derived cell culture material of the present invention is comply with the medical aseptic requirement.
The soft tissue filler composition for injection of the present invention being confirmed its safety as described above was a light white suspension. As
a result of BrDU antibody test (Huang hui (««¥) , Lai Xinan (*K P3 rø ) }
Wang Zhengguo (UEH) , Wang LiIi (ΞE UU) , Roles of a material during the wound healing in transferring to dermal stem cells and delivering a receptor; Chinese Journal of Trauma, 2004, 20(3): 142-145), the occupying ratio of the dermal fibroblast stem cells in the filler composition was in the range from 10 to 20%. The cell morphological observation showed that the occupying ratios of the dermal fibroblast transit amplifying cells and dermal fibroblasts are in the range from 30 to 40% and 50 to 70%, respectively. Further, as a result of measuring the amount of collagen according to the method as described in the literature (Ministry of Health P.R.China, Pharmacopoeia Committee, <Chinese Pharmacopoeia> (2nd Ed.), Beijing Chemical Industry Press, 2000), the content of collagen in the filler composition of the present invention was in the range from 20 to 60 mg/ml.
The soft tissue filler composition for injection of the present invention was stored and transported at 4°C by using a specific storage box, and stored under the environmental condition where is cool and dry, capable of keeping out of the direct exposure to the sun, and free from corrosive gas and high pressure.
<Example 2> Therapeutic effect of a soft tissue filler on the removal of wrinkles
Therapeutic effect of the soft tissue filler composition for injection prepared in Example 1 on the removal of wrinkles was clinically examined as follows.
The patients to be treated, who are 60 years old or below and suffered from facial wrinkles such as forehead wrinkles, glabellar furrows (the space between the eyebrows and above the nose), nasolabial folds, marrionette lines and the like, were volunteered for this clinical test. Among the volunteers, the patients who have autoimmune disease, chronic cutaneous disorder, communicable disease, acute and chronic infectious disease, infection on the wrinkled area, pregnancy, severe heart, liver and kidney diseases, sensitive constitution and the like were ruled out. The finally selected 20 patients were subjected to blood and urine tests, an electrocardiogram, tests for liver and kidney function, tests for the presence of HIV and HBs Ag and the like.
The wrinkled area of the patient was sterilized with 70% alcohol, followed by anesthetizing the dermis thereof by injecting 1% lidocaine. 1.5 mi of the soft tissue filler composition prepared in Example <1-1> was filled into a 3 i! syringe equipped with a 4.5 needle in length of 2.2 cm. The needle was pricked to several points on the anesthetized dermis tilted to one side and then the composition was injected between the upper layer and the middle layer of the dermis. At this time, the angle of the syringe needle to the injection area on the skin was maintained at a range from 20 to 45°. During the treatment, the skin was drawn to the full until turn pale and left to make room on the injection area. After the treatment, the injection area was massaged with an ice bag for 2 hours. A total of three injections were repeated at the same injection dose once every two weeks as described above. Then, the injection area was monitored for 12 months.
The presence of abnormal symptoms on the injection area was examined by observing local or systemic conditions of the patient and the patients were taken vitamin C twice every days (200 mg per once, 400 mg/day) for 6 months. It was noticed to the patients that the injection area must be prevented from directly exposing to the sun and contacting with irritable cosmetics within 3 days after the treatment.
The effect on wrinkle improvement was estimated according to the following standards: Excellent: wrinkles are eliminated and the skin is completely recovered.
Good: wrinkles are mitigated and clearly improved. Poor: wrinkles are not improved.
As a result of observing the facial wrinkles after the treatment, the therapeutic effects at the time of 3, 6, 9 and 12 months after the treatment were
68.3%, 79.2%, 88.1% and 91.7%, respectively. The therapeutic ratio for each wrinkled area on the face was 91.4% for wrinkles, 94.9% for fine wrinkles, 93% for glabellar furrows, 88.2% for the philtrum, and 88.9% for nasolabial folds and marrionette lines, and the mean time of lasting the therapeutic effects was 3.5 months. All the treated patients showed significantly higher satisfaction in a statistical analysis than a control. Further, there was no meaningful side effect except transient skin flare on the injection area.
<Example 3> Therapeutic effect of a soft tissue filler on the removal of scars
The soft tissue filler composition for injection was prepared according to the same method as described in Example <1-1>, except that the cell subculture was conducted for 8 weeks. Thus prepared filler composition contained autologous dermis-derived cells at a concentration ranging from 3x 107 to 7x 107 cells/m^ as an effective ingredient.
The safety of the soft tissue filler composition prepared above was confirmed by performing tests for viral infection and immune response and aseptic test according to the same methods as described in Example <l-2>.
Further, as a result of examining cell distribution of the autologous dermis-derived cells in the soft tissue filler composition prepared by culturing for 8 weeks, the occupying ratio of dermal fibroblast stem cells was in the range from 15 to 20%, that of dermal fibroblast transit amplifying cells was in the range from 20 to 50%, and that of dermal fibroblasts was in the range from 30 to 50%. In addition, the content of collagen in the filler composition was in the range from 30 to 80 mg/m#.
Therapeutic effect of the soft tissue filler composition for injection prepared in Example 1 on the removal of scars was clinically examined as follows.
Twenty volunteers having scars caused by facial acne or wound were selected according to the same conditions and subjected to the pretests according to the same methods as described in Example 2. Thus selected subjects were treated with the soft tissue filler composition at the scar area once every two weeks. A total of three injections were repeated at the same dose for 6 weeks as described in Example 2 and the injection area was monitored for 12 months after the injection. At this time, the effect on scar improvement was estimated according to the following standards: Excellent: scars are eliminated and the skin is completely recovered.
Good: scars are mitigated and clearly improved. Poor: scars are not improved. As a result, the therapeutic effect on the removal of scars at the time of 3, 6, 9 and 12 months after the treatment were 67.5%, 92.5%, 92.5% and 92.5%, respectively, and the mean time of lasting this therapeutic effects was 3.4 months. All the treated patients showed significantly higher satisfaction in a statistical analysis than a control. Further, there was no meaningful side effect except transient skin flare on the injection area.
<Example 4> Skin texture improving and cosmetic effects of a soft tissue filler
The soft tissue filler composition for injection was prepared according to the same method as described in Example <1-1>, except that the cell subculture was conducted for 8 weeks. Thus prepared filler composition contained autologous dermis-derived cells at a concentration ranging from 1.5x 107 to 5.5 x 107 cells/m£ as an effective ingredient.
The safety of the soft tissue filler composition prepared above was confirmed by performing tests for viral infection and immune response, and aseptic test according to the same methods as described in Example <l-2>. Further, as a result of examining cell distribution of the autologous dermis-derived cells in the soft tissue filler composition prepared by culturing for 8 weeks, the occupying ratio of dermal fibroblast stem cells was in the range from 5 to 20%, that of dermal fibroblast transit amplifying cells was in the range from 20 to 50%, and that of dermal fibroblasts was in the range from 30 to 70%. Additionally, the content of collagen in the filler composition was in the range from 30 to 60 mg/mH.
Skin texture improving and cosmetic effects of the soft tissue filler composition for injection prepared in Example 1 were clinically examined as follows. Twenty volunteers having coarse and wrinkled skin texture and dull skin tone or who wish to augment the lips or the philtrum (the groove between the upper lip and the nose) were selected according to the same conditions and subjected to the pretests according to the same methods as described in Example 2. Thus selected subjects were treated with the soft tissue filler composition at the target area once every two weeks. A total of three injections were repeated at the same dose for 6 weeks as described in Example 2, and the injection area was monitored for 12 months after the injection. As a result, it has been confirmed that the treated patient's skin becomes smoothed and even, fine wrinkles thereon are eliminated. Further, skin resilience is recovered with being glossy at the time of 3 months from the treatment. Also, the lips were clearly refined and augmented, and the groove between the upper lip and the nose was flattened.
<Comparative Example 1>
A soft tissue filler composition for injection for removing wrinkles and scars was prepared according to the method described in Chinese Patent Application No. 03155833.X, and its therapeutic effect on the removal of wrinkles and scars was examined. In particular, the filler composition was prepared according to the same method as described in Example <1-1>, except that cells were cultured in a dermal cell culture medium DMEM (D5546, Sigma, USA) supplemented with fetal bovine serum for 5 weeks. Thus prepared filler composition contained cells at a concentration ranging from I x IO7 to 2x 107 cells/mβ as an effective ingredient.
The safety of the soft tissue filler composition prepared above was confirmed by performing tests for viral infection and immune response, and aseptic test according to the same methods as described in Example <l-2>. As a result of examining cell distribution of the cells in the soft tissue filler composition prepared by culturing for 5 weeks, the occupying ratio of dermal fibroblast stem cells was in the range from 5 to 15%, that of dermal fibroblast transit amplifying cells was in the range from 30 to 50%, and that of dermal fibroblasts was in the range from 40 to 60%. Additionally, the content of collagen in the filler composition was in the range from 2 to 5 mg/m#.
In order to examine the therapeutic effect on the removal of wrinkles and scars of the soft tissue filler composition prepared above, a clinical test was conducted for twenty-eight volunteers according to the same method as described in Example 2. As a result, it has been found that the filler composition shows clear therapeutic effects on the removal of wrinkles and scars only from the time of 6 to 9 months after the treatment. From 6 months after the treatment, fine wrinkles at the corner of the eyes became eliminated, forehead lines got smaller or disappeared, and skin texture and resilence were improved.
Further, to confirm that the cells included in the autologous dermis- derived cell culture material prepared according to the method of the present invention are neural progenitor-derived cells showing the characteristics specific for dermis-derived fibroblasts that are discriminated from mesenchymal-derived stem cells, the following experiments were carried out by using adipose tissue- derived cells as a comparative control.
<Example 5> Morphological comparison of dermis-derived cells with adipose tissue-derived cells
To obtain the dermis-derived cell culture material according to the present invention, a tissue sample was collected from the back of the ear in a size of 3 to 4 mnf. This tissue sample was subjected to tissue digestion by adding a solution of pancreatin/EDTA, which was stopped by adding DMEM supplemented with 10% FBS. The digested tissue sample was cut into pieces and broken into single cells by pipetting several times and centrifuged to remove a supernatant, thereby recovering only a cell pellet. Thus separated cell pellet was re-suspended in a serum-free culture medium and cultured in a 5% CO2 incubator at 370C . At this time, the serum- free culture medium was prepared by adding 1 g/m# of hydrocortison, 10 ng/mi of hEGF, 3 ng/m-β of bFGF and 10 g/m£ of heparin to a fibroblast culture basal medium (DMEM or 106; Cascade). Cell morphology was observed with a phase-contrast microscope (Olympus IX 71) at the time of 3 -week after the cultivation and recorded with a digital camera system.
Meanwhile, adipose tissue-derived stem cells were obtained by the following procedure, i.e., a fat suction solution obtained after the liposuction was subjected to lysis by treating with 0.1% collagenase at 37°C for 45 minutes followed by centrifuging the resulting solution to remove a supernatant, thereby separating only a cell pellet. Thus separated cell pellet was re-suspended in DMEM supplemented with 10% FBS and cultured in a 5% CO2 incubator at 37 °C . Cell morphology was observed with a phase-contrast microscope (Olympus IX 71) at the time of 3 -week after the cultivation and recorded with a digital camera system.
As a result, as shown in Fig, 4, while the dermis-derived cells according to the present invention showed a fibroblast-like longish shape, the adipose tissue-derived cells showed a flatly stretched shape in all directions. Such a morphology of each cell coincided with that previously reported in the literature (Patricia A. ZUK et al., Tisseu Engineering 7: 211-228, 2001; and Patricia A. ZUK et al., Molecular Biology of the Cell 13: 4279-4295, 2002). <Example 6> Immunological comparison of dermis-derived cells with adipose tissue-derived cells
<6-l> Immunofluorescence analysis using nestin and fibronectin as a marker
The dermis-derived cells were cultured in a serum-free culture medium for 2 weeks according to the same method as described in Example 5. When the confluence of the dermis-derived cells reaches 90%, 0.25% trypsin/EDTA solution was added to the culture solution to detach the cells from the culture plate, followed by re-suspending the cells in a serum-free culture medium. Thus prepared cell suspension was smeared onto a slide glass and incubated in a CO2 incubator at 37 "C for 12 to 16 hours. When the cells were proliferated at the proper concentration, the slide glass was washed with DPBS, dried and then used as a sample for the following immunofluorescence analysis. Further, the adipose tissue-derived cells were cultured in DMEM supplemented with 10% FBS according to the same method as described in Example 5. When the confluence of the adipose tissue-derived cells reaches 90%, 0.25% trypsin/EDTA solution was added to the culture solution to detach the cells from the culture plate, followed by re-suspending the cells in DMEM supplemented with 10% FBS. Thus prepared cell suspension was smeared onto a slide glass and incubated in a CO2 incubator at 37 °C for 12 to 16 hours. When the cells were proliferated at the proper concentration, the slide glass was washed with DPBS, dried and then used as a sample for the following immunofluorescence analysis. Each slide glass prepared above was soaked in 3.7% paraformaldehyde in PBS as a solvent for 10 minutes under shaking to fix the cells and washed with 1% skim milk in PBS for 10 minutes. Each slide glass was then soaked in 0.5% triton X-100 in PBS for 10 minutes under shaking to enhance cell permeability, followed by washing with 1% skim milk in PBS for 10 minutes. Nestin (Anti-Rabbit Polyclonal Antibody, CHEMICON) and
Fibronectin (Mouse Anti-Fibronectin Polyclonal Antibody, Santa cruz) were diluted with 1% skim milk in PBS by 1 :500, respectively. Each of the diluted marker solutions was added to the slide glass and reacted for 1 hour under shaking. Thereafter, the slide glass was washed three times with 1 % skim milk in PB S for 10 minutes .
Alexa 594 (Anti-Rabbit IgG Antibody, Molecular probe) and Alexa 488 (Anti-Mouse IgG Antibody, Molecular probe) were diluted with 1% skim milk in PBS by 1 :500, respectively. Each of the diluted antibody solutions was added to the slide glass and reacted for 30 minutes under shaking.
DAPI (4',6-Diamidin-2-phenylindole dihydrochloride, SIGMA) was diluted with PBS by 1 :20000. The slide glass was then soaked in the DAPI solution to stain the cells and washed three times with DPBS for 10 minutes.
The washed slide glass was observed with a fluorescent microscope (Olympus
IX 71) and recorded with a digital camera system.
As a result, as described in Fig. 5, nestin and fϊbronectin were expressed in the dermis-derived cells according to the present invention. The cells expressing both of them were then observed. However, in the adipose tissue- derived cells, the expression of fibronectin was detected, but nestin was not expressed. These results have confirmed that since the dermis-derived cells cultured according to the method of the present invention exhibit a different expression pattern of cell markers from the adipose tissue-derived cells and display nestin (a marker for neural progenitor cells), they show immunological characteristics corresponding to neural progenitor-derived stem cells and not mesenchymal-derived stem cells (Toma et al., Stem Cells 23: 727-737, 2005; Fernandes et al., Nature 6: 1082-1093, 2004; and Toma et al., Nature 3: 778- 784, 2001).
<6-2> Immunofluorescence analysis using nestin and vimentin as a marker The slide glass samples of the dermis-derived cells and adipose tissue- derived cells for the immunofluorescence analysis were prepared according to the same method as described in Example <6-l>, respectively, and treated with paraformaldehyde to fix the cells, followed by treating with triton X-100 to improve cell permeability.
Nestin (Anti-Rabbit Polyclonal Antibody, CHEMICON) and Vimentin (Mouse Anti- Vimentin Monoclonal Antibody, CHEMICON) were diluted with 1% skim milk in PBS by 1 :500, respectively. Each of the diluted marker solutions was added to the slide glass and reacted for 1 hour under shaking. Thereafter, the slide glass was washed three times with 1% skim milk in PBS for 10 minutes.
Alexa 594 (Anti-Rabbit IgG Antibody, Molecular probe) and Alexa 488 (Anti-Mouse IgG Antibody, Molecular probe) were diluted with 1% skim milk in PBS by 1 :500, respectively. Each of the diluted antibody solutions was added to the slide glass and reacted for 30 minutes under shaking.
DAPI (4',6-Diamidin-2-phenylindole dihydrochloride, SIGMA) was diluted with PBS by 1 :20000. The slide glass was then soaked in the DAPI solution to stain the cells and washed three times with DPBS for 10 minutes. The washed slide glass was observed with a fluorescent microscope (Olympus IX 71) and recorded with a digital camera system. As a result, as illustrated in Fig. 6, nestin and vimentin were expressed in the dermis-derived cells of the present invention. Then, the cells expressing both of them were observed. However, in the adipose tissue-derived cells, the expression of vimentin was detected, but nestin was not expressed. These results were identical to the previous report as described in the literature (Toma et al, Stem Cells 23: 727-737, 2005; Fernandes et al., Nature 6: 1082-1093, 2004).
<6-3> Immunofluorescence analysis using nestin and fibroblast surface protein 1 (FSPl) as a marker The slide glass samples of the dermis-derived cells and adipose tissue- derived cells for the immunofluorescent analysis were prepared according to the same method as described in Example <6-l>, respectively, and treated with paraformaldehyde to fix the cells, followed by treating with triton X-100 to improve cell permeability. FSPl (Monoclonal Anti-Human Fibroblast Surface Protein Clone IB lO,
SIGMA) was diluted with 1% skim milk in PBS by 1 :500 and added to the slide glass. The slide glass was reacted for 1 hour under shaking. Thereafter, the slide glass was washed three times with 1% skim milk in PBS for 10 minutes.
After the washing, each slide glass was soaked in 3.7% paraformaldehyde in PBS for 10 minutes under shaking to fix the cells and washed with 1% skim milk in PBS for 10 minutes. The slide glass was soaked in 0.5% triton X-100 in PBS for 10 minutes to increase cell permeability, followed by washing with 1% skim milk in PBS for 10 minutes.
Nestin (Anti-Rabbit Polyclonal Antibody, CHEMICON) was diluted with 1% skim milk in PBS by 1 : 1000 and added to the slide glass. The slide glass was reacted for 1 hour under shaking and then washed three times with 1% skim milk in PBS for 10 minutes.
Alexa 594 (Anti-Rabbit IgG Antibody, Molecular probe) and Alexa 488 (Anti-Mouse IgG Antibody, Molecular probe) were diluted with 1% skim milk in PBS by 1:500, respectively. Each of the diluted antibody solutions was added to the slide glass and reacted for 30 minutes under shaking.
DAPI (4',6-Diamidin-2-phenylindole dihydrochloride, SIGMA) was diluted with PBS by 1 :20000. The slide glass was then soaked in the DAPI solution to stain the cells and washed three times with DPBS for 10 minutes. The washed slide glass was observed with a fluorescent microscope (Olympus IX 71) and recorded with a digital camera system. As a result, as described in Fig. 7, nestin and FSPl were expressed in the dermis-derived cells of the present invention. Then, the cells expressing both of them were observed. However, in the adipose tissue-derived cells, the expression of FSPl was detected, but nestin was not expressed.
It has been confirmed from these results that the dermis-derived cell culture material obtained by culturing in a serum-free medium according to the present invention shows signals for fibronectin and FSP-I known as a fibroblast marker protein as well as for nestin known as a neural progenitor-derived stem cell marker protein, which are clearly different from mesenchymal-derived stem cells such as adipose tissue-derived cells.
<Example 7> RT-PCR for examining the expression pattern of neural progenitor marker proteins in dermis-derived cells
The dermis-derived cells were cultured in a serum-free culture medium for 2 weeks according to the same method as described in Example 5. When the confluence of the dermis-derived cells reaches 90%, 0.25% trypsin/EDTA solution was added to the culture solution to detach the cells from the culture plate, followed by RNA extraction by using a RNA extraction kit (Purelink Micro to-midi, Invitrogen).
A reaction solution was prepared by mixing thus extracted RNA, dNTP and oligo-dT 20 mer primer and adjusting its final volume to 10 μJt. The reaction solution was incubated at 65 °C for 5 minutes, followed by keeping at 0°C for a minute. After 10 μi of a cDNA synthetic mixture (1OxRT buffer, 25 mM MgCl2, 0.1 M DTT, RNase OUT, Superscript IH RT) was added thereto, the resulting solution was subjected to reverse transcription at 50 °C for 50 minutes and 85 °C for 20 minutes. To the reaction solution was added 1 μJt of RNAse H and kept at 37°C for 20 minutes to thereby synthesize cDNA.
PCR was carried out by using the synthesized cDNA as a template to examine the expression patterns of p75NTR, Pax3, Snail, Slug, nestin and vimentin known as a neural progenitor marker protein. The PCR conditions were as follows, i.e., initial denaturation at 95 °C for 2 minutes, followed by 35 cycles of 95 °C for 30 seconds, 55 °C for 30 seconds and 72 °C for 1 minute. At this time, GAPDH was used as a control, and forward and reverse primer pairs used in the PCR for the amplification of each marker protein were as follows:
SEQ ID NO: 1 of nestin-1 and SEQ ID NO: 2 of mestin-2 for nestin amplification
SEQ ID NO: 3 of vimentin-1 and SEQ ID NO: 4 of vimentin-2 for vimentin amplification
SEQ ID NO: 5 of p75NTR-l and SEQ ID NO: 6 of p75NTR-2 for p75NTR amplification
SEQ ID NO: 7 of pax3-l and SEQ ID NO: 8 of ρax3-2 for Pax3 amplification SEQ ID NO: 9 of snail- 1 and SEQ ID NO: 10 of snail-2 for Snail amplification
SEQ ID NO: 11 of slug-1 and SEQ ID NO: 12 of slug-2 for Slug amplification
SEQ ID NO: 13 of GAPDH-I and SEQ ID NO: 14 of GAPDH-2 for GAPDH amplification
As a result, as illustrated in Fig. 8, it has been confirmed that p75NTR, Pax3, Snail, Slug, nestin and vimentin are expressed at the mRNA level in the dermis-derived cells of the present invention. In particular, vimentin was abundantly expressed in the dermis-derived cells of the present invention. These results demonstrated the fact at the mRNA level that the dermis-derived cell culture material of the present invention contains neural progenitor-derived stem cells.
Industrial Applicability
As described above, the soft tissue filler composition for injection of the present invention exerts prolonged therapeutic effects without causing any immune response and side-effect due to the use of autologous dermal cells. Further, since it completely rules out the risk of the use of animal-derived serum through in vitro serum-free cultivation and immediately shows therapeutic effects by producing a large quantity of collagen, the soft tissue filler composition for injection of the present invention can be effectively used for improving the skin tone and resilience as well as smoothing and removing wrinkles and scars.

Claims

What is claimed is:
1. A method of preparing a soft tissue filler composition for injection, comprising the steps of:
1) digesting an autologous dermal tissue isolated from the patient's own skin and separating into single cells;
2) culturing and proliferating the isolated dermal cells via in vitro serum-free cultivation to obtain an autologous dermis-derived cell culture material containing dermal fibroblast stem cells, dermal fibroblast transit amplifying cells, dermal fibroblasts and collagen; 3) centrifuging the autologous dermis-derived cell culture material to separate an autologous dermis-derived cell pellet; and
4) suspending the autologous dermis-derived cell pellet in a glucose injection or a random injection to obtain a suspension for injection.
2. The method according to Claim 1, wherein the autologous dermal tissue in Step 1) is to be thawed once after a dermal tissue isolated from the patient's own skin is lyophilized and stored.
3. The method according to Claim 1, wherein the soft tissue filler composition comprises l * 107 to 8x 107 cells/m-C of autologous dermis-derived cells and 10 to 100 mg/ml of collagen as an effective ingredient.
4. The method according to Claim 1 , wherein the ratio of dermal fibroblast stem cells, dermal fibroblast transit amplifying cells and dermal fibroblasts in the soft tissue filler composition is 5 to 20%: 20 to 50%: 30 to 70%.
5. The method according to Claim 1, wherein the medium for in vitro serum- free cultivation contains a growth factor and an activation factor.
6. The method according to Claim 5, wherein the growth factor is one or more selected from the group consisting of epidermal growth factor, dermal growth factor, bFGF (basic fibroblast growth factor) and a mixture thereof.
7. The method according to Claim 6, wherein the content of the growth factor is in the range from 0.1 to 1 ng/vd.
8. The method according to Claim 5, wherein the activation factor is one or more selected from the group consisting of cortical hormone, bovine pituitary extract (BPE), insulin, hydrocortisone and a mixture thereof.
9. The method according to Claim 8, wherein the content of the activation factor is in the range from 0.1 to 1%.
10. The method according to Claim 1, wherein the in vitro serum- free cultivation is conducted for 6 to 10 weeks.
11. A soft tissue filler composition for injection for alleviating or treating skin damage due to mechanical or physiological causes, which is prepared by the method according to claim 1.
12. The soft tissue filler composition for injection according to Claim 11 , which is intended for alleviating or treating wrinkles or scars at the area having skin damage due to dermal defects.
13. The method according to Claim 12, wherein the treatment of skin damage due to dermal defects is intended for reducing wrinkles, removing scars or augmenting the lips.
14. The method according to Claim 13, wherein the wrinkles include wrinkles on the face and neck, stretch marks from pregnancy and wrinkles on the back of the hand.
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