EP2049130A1 - Soft tissue filler composition comprising autologous dermis-derived cell culture product and hyaluronic acid - Google Patents

Soft tissue filler composition comprising autologous dermis-derived cell culture product and hyaluronic acid

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Publication number
EP2049130A1
EP2049130A1 EP07747120A EP07747120A EP2049130A1 EP 2049130 A1 EP2049130 A1 EP 2049130A1 EP 07747120 A EP07747120 A EP 07747120A EP 07747120 A EP07747120 A EP 07747120A EP 2049130 A1 EP2049130 A1 EP 2049130A1
Authority
EP
European Patent Office
Prior art keywords
soft tissue
dermal
cells
filler composition
tissue filler
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP07747120A
Other languages
German (de)
French (fr)
Other versions
EP2049130A4 (en
Inventor
Bin Han
Jay Do Choi
Min Jung Nam
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
S-biomedics Co Ltd
Original Assignee
S-biomedics Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by S-biomedics Co Ltd filed Critical S-biomedics Co Ltd
Publication of EP2049130A1 publication Critical patent/EP2049130A1/en
Publication of EP2049130A4 publication Critical patent/EP2049130A4/en
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • 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
    • 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
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/0059—Cosmetic or alloplastic implants
    • 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

  • the present invention relates to a soft tissue filler composition for injection comprising an autologous dermis-derived cell culture material and hyaluronic acid which is useful for alleviating and removing wrinkles and scars.
  • 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 19 th century. However, it gave rise to too many side effects and its filling effect was not satisfactory. Approximately twenty years ago, a method of employing bovine collagen for the treatment of wrinkles and scars was developed. However, bovine collagen induced allergic and heterologous rejection reactions to the patients being treated. Further, once implanted, the collagen is rapidly absorbed back into the tissue within a short time and its therapeutic effects can be maintained only for a few months.
  • Fibrel the source of which is porcine collagen
  • Artecoll is a complex of artificially synthesized microspores and bovine collagen. Artecoll exhibits prolonged therapeutic effects by providing autologous collagen through the stimulation of regional dermis.
  • the subject being treated can feel the presence of the particles and the swelling may occur at the treatment site.
  • 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 fibrocytes.
  • 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.
  • 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.
  • non-animal stabilized hyaluronic acid available under the trade name RestylandTM (Q-Med Scandinavia Inc., USA) was approved by FDA on April, 2002 as an injection for smoothening wrinkles and augmenting the lips.
  • Hyaluronic acid is a naturally-occurring linear polysaccharide with repeating disaccharide units composed of gluconic acid and N-acetyl-glucosamine, It functions to provide nutrients to the tissue and cells simultaneously with offering liquid environment useful for tissue metabolism and makes the skin resilient and smooth.
  • the hyaluronic acid is employed as soft tissue filler, it may be advantageous in that allergic reactions and side-effects may be avoided since it is a natural substance.
  • the hyaluronic acid may be disadvantageous since the duration of therapeutic effect is too short.
  • a soft tissue filler composition comprising an autologous dermis-derived cell culture material, which is prepared by separating from the patient's own skin and proliferating via in vitro serum-free cultivation, and hyaluronic acid as an effective ingredient shows continuous natural and good therapeutic effects.
  • a composition can be effectively used for removing wrinkles and scars.
  • the primary object of the present invention is to provide a soft tissue filler composition showing immediate and continuous remarkable therapeutic effects for treating skin damage due to dermal defects.
  • a soft tissue filler composition for injection which comprises an autologous dermis-derived cell culture material and hyaluronic acid as an effective ingredient, wherein the autologous dermis-derived cell culture material is prepared by separating from the patient's own skin and proliferating via in vitro serum- free cultivation.
  • the autologous dermis-derived cell culture material of the present invention may be obtained by separating a dermal biopsy from the patient's own skin and proliferating the same via in vitro serum-free cultivation. Such material is a complex of dermis-derived cells containing dermal fibroblast stem cells, dermal fibroblast transit amplifying cells and dermal fibroblasts.
  • the autologous dermis-derived cell culture material of the present invention may further comprise collagen obtained together with said dermis-derived cells during the in vitro serum-free cultivation.
  • the dermal fibroblast stem cells, dermal fibroblast transit amplifying cells, dermal fibroblasts and collagen in the filler composition are derived from a small amount of the patient's own skin (1 to 30 mm 2 ).
  • an autologous dermal biopsy isolated from the patient's own skin is digested and separated into single cells. Then, thus separated single dermal cells are subjected to in vitro cultivation and proliferation in an in vitro serum- free culture medium, thereby obtaining an autologous dermis-derived cell culture material containing dermal fibroblast stem cells, dermal fibroblast transit amplifying cells, dermal fibroblasts and collagen as a complex filler.
  • the autologous dermis-derived cell culture material in the soft tissue filler composition of the present invention may be prepared by the following steps:
  • the autologous dermis-derived cell material obtained according to the method of the present invention is composed of dermis-derived cells containing dermal fibroblast stem cells, dermal fibroblast transit amplifying cells and dermal fibroblasts proliferated by in vitro serum-free cultivation, as well as collagen secreted therefrom.
  • the total number of cells as an effective ingredient in the cell culture material is preferably in the range from I x IO 7 to 8x 10 7 cells/m£, more preferably I x IO 7 to 5 ⁇ lO 7 cellsM, while the content of collagen therein is preferably in the range from 10 to 100 mg/m£, more preferably 20 to 60 mg/m#.
  • 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 mnf 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.
  • the autologous dermal tissue sample is subjected to tissue digestion and cell isolation procedures.
  • 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.
  • 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.
  • 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.
  • growth factors e.g., epidermal growth factor and dermal growth factor
  • activation factors e.g., cortical hormone and bovine pituitary extract
  • 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.
  • the content of collagen in the filler may range from 10 to 100 mg/ml
  • 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/mt.
  • 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% 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.
  • 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 autologous dermis-derived cell culture material thus prepared contains dermal fibroblast stem cells, dermal fibroblast transit amplifying cells, dermal fibroblasts and collagen as an effective ingredient, wherein the mixed ratio of the dermal fibroblast stem cells, dermal fibroblast transit amplifying cells and dermal fibroblasts is 5 to 20% : 20 to 50% : 30 to 70%, while the content of collagen is in the range from 10 to 100 mg/m#, preferably 20 to 60 mg/m-£.
  • Hyaluronic acid useful in the present invention may include natural hyaluronic acid isolated from human skin or an animal source and modified hyaluronic acid produced by fermenting genetically engineered microorganisms, e.g., a hyaluronic acid product commercially produced by Shandong Freda Biochem Co., Ltd.
  • Hyaluronic acid is a glycosaminoglycan heteropolysaccharide distributed widely throughout connective, epithelial and neural tissues. It is one of the chief components of the extracellular matrix between epidermal and dermal layers. It is more effective to inject hyaluronic acid having a higher molecular weight for restoring a depressed skin area.
  • hyaluronic acid having a molecular weight of 5,000,000 daltons or more has extremely strong viscosity to use as an injection, it is preferable to employ hyaluronic acid having a molecular weight ranging from 1,000,000 to 5,000,000 daltons.
  • the soft tissue filler composition of the present invention contains hyaluronic acid at a concentration ranging from 2 to 20 wg/mH, preferably 5 to 15 mg/ml
  • the soft tissue filler composition for removing wrinkles and scars may be prepared by obtaining the autologous dermis-derived cell culture material containing dermal fibroblast stem cells, dermal fibroblast transit amplifying cells, dermal fibroblasts and collagen through the in vitro serum-free cultivation of the patient's own dermal biopsy and mixing it with 1 to 4 ml of hyaluronic acid at a concentration ranging from 2.0 to 20 mg/m ⁇ so as to adjust the final cell concentration to Ix IO 7 to 8* 10 7 cells/ml
  • hyaluronic acid is a natural substance, it is safe and does not cause any allergic reaction or side-effect. Thus, it can be effectively used as an epidermal layer filling material. However, its therapeutic effect lasts only for 6 months.
  • the removal of wrinkles using only autologous dermis-derived cells can safely improve facial wrinkles or depressed scars through the prolonged therapeutic effect, thereby highly satisfying a patient being treated.
  • the present invention is characterized by mutually complementing the problems caused by the single use of the autologous dermis-derived cells or hyaluronic acid alone through the combination thereof, thereby achieving the purpose of the immediate onset and prolonged maintenance of therapeutic effect.
  • 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.
  • 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.
  • suitable examples of an excipient include, but are not limited to, phosphate buffered saline, bacteriostatic saline, propylene glycol, starch, sucrose and sorbitol.
  • 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.
  • 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 of the present invention comprises the autologous dermis-derived cell culture material originated from the patient's own skin as an effective ingredient, there is no risk of causing any rejection reaction or side-effect. Further, it can keep up its prolonged therapeutic effect for several years. Also, the injection of the present invention can bring an immediate onset of therapeutic effect due to hyaluronic acid among the dose unit, while the short-term therapeutic effect of hyaluronic acid is complemented by the autologous dermis-derived cells showing therapeutic effects only after 3 to 9 months have passed from the onset of the treatment. This causes the therapeutic effects of the injection to last for a long time. Accordingly, the injection of the present invention has advantages of immediately exerting remarkable filling and restoring effects after application to a target site as well as continuously maintaining such a therapeutic effect for a long time.
  • hyaluronic acid used in the injection of the present invention provides a favorable environment for the growth of autologous dermis-derived cells included therein, it can be expected to further produce collagen as these cells grow continuously after the injection. That is, since the injection of the present invention produces additional collagen at the injection site after the treatment besides the collagen originally contained therein, it shows excellent therapeutic effects of smoothening and removing the wrinkles and scars. Further, it is capable of improving the local environment of the skin, which can be effectively used for restoring natural and more youthful appearance in case of applying to the face.
  • the injection of the present invention exhibits better and more natural therapeutic effects and can maintain the same for a long time compared to injections of bovine collagen, autologous collagen and Botox.
  • the present invention further comprises a method of alleviating or removing wrinkles or scars by injecting the injection of the present invention to the area having skin damage due to dermal defects, e.g., wrinkles or scars.
  • the method can be effectively used for alleviating or removing wrinkles on the face and neck, stretch marks from pregnancy or wrinkles on the wrist and augmenting the lips.
  • the soft tissue filler composition comprising an autologous dermis- derived cell culture material and hyaluronic acid as an effective ingredient according to the present invention shows the complementary action of both hyaluronic acid responsible for a rapid onset of therapeutic effect and the autologous dermis-derived cell culture material responsible for a long-term maintenance thereof without causing any immune response and side-effect. Accordingly, it can be effectively used for smoothening and removing the wrinkles and scars as well as improving the skin tone and resilience.
  • Fig. 1 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. 2 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) 5 wherein nestin is detected as a red spot at 594 nm, fibronectin is detected as a green spot at 488 run and cell nuclei is detected as a blue spot by DAPI staining;
  • Fig. 3 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.
  • FIG. 4 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. 5 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.
  • RT-PCR reverse transcription-polymerase chain reaction
  • the epidermal and dermal tissue sample was excised in a size of 4 mnf 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 mi of 0.05% pancreatin/EDTA solution was added thereto, and then the culture dish was kept in a CO 2 incubator at 37 ° C for 10 minutes to induce cell digestion.
  • the digestion reaction was completed by adding 5 mi 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 rpm/min for 5 minutes to remove a supernatant, thereby recovering only a cell pellet.
  • a dermal cell culture medium (Cascade, Cat. No. 106, supplemented with 1 ng/mi 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 CO 2 incubator at 37 °C under 5% CO 2 .
  • 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 ⁇ 10 7 to 6x 10 7 cells/m ⁇ as an effective ingredient.
  • Example ⁇ 1-1> 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 mi 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.
  • 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 mi of the cell culture material.
  • 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.
  • aseptic test according to Chinese Biological Product Regulation (2000 Ed.), General Principle ⁇ Biological Product Aseptic Test Regulation ⁇ Article AfB, 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.
  • 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 (age ranging from 18 to 60) who are 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.
  • 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 11 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 ml of the soft tissue filler composition prepared in Example ⁇ 1-1> was filled into a 3 m£ 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 ° .
  • 15 or 20 injection points were taken at the wrinkled area, while 0.05 to 0.1 mi of the filler composition was injected into one point.
  • the skin was drawn to the full until turn pale and left to make room on the injection area.
  • the injection area was massaged with an ice bag for 2 hours. Within 24 hours after the treatment, light swelling or tingling at the injection area may have occurred. However, there was no local or systemic symptom in the entire body. 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 therapeutic effect of hyaluronic acid on the wrinkle improvement was immediately shown in the patient treated with the injection comprising the autologous dermis-derived cell culture material and hyaluronic acid as an effective ingredient according to the present invention.
  • the dermal fibroblast transit-amplifying cells in the autologous dermis-derived cell culture material were gradually matured and converted into the dermal fibroblasts with producing autologous collagen in the course of time, which makes the thickness and density of the dermal layer to be increased, wrinkles and depressed scars to be improved, and the skin tone and resilience to be restored. Said therapeutic effects were maintained for a long time.
  • 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.
  • tissue sample was collected from the back of the ear in a size of 3 to 4 m ⁇ f. 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% CO 2 incubator at 37 0 C .
  • the serum-free culture medium was prepared by adding 1 g/m£ of hydrocortison, 10 ng/ml of hEGF, 3 ng/iM 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.
  • DMEM or 106 fibroblast culture basal medium
  • 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 0 C for 45 minutes followed by centrifuging the resulting solution to remove a supernatant, thereby separating only a cell pellet.
  • a fat suction solution obtained after the liposuction was subjected to lysis by treating with 0.1% collagenase at 37 0 C for 45 minutes followed by centrifuging the resulting solution to remove a supernatant, thereby separating only a cell pellet.
  • DMEM fetal bovine serum
  • FBS fetal bovine serum
  • the dermis-derived cells were cultured in a serum-free culture medium for 2 weeks according to the same method as described in Example 3.
  • 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.
  • prepared cell suspension was smeared onto a slide glass and incubated in a CO 2 incubator at 37 ° C for 12 to 16 hours.
  • the slide glass was washed with DPBS, dried and then used as a sample for the following immunofluorescence analysis.
  • adipose tissue-derived cells were cultured in DMEM supplemented with 10% FBS according to the same method as described in Example 3.
  • 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.
  • prepared cell suspension was smeared onto a slide glass and incubated in a CO 2 incubator at 37 ° C for 12 to 16 hours.
  • 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-IOO in PBS for 10 minutes under shaking to enhance cell permeability, followed by washing with 1% skim milk in PBS for 10 minutes.
  • Alexa 594 Anti-Rabbit IgG Antibody, Molecular probe
  • Alexa 488 Anti-Mouse IgG Antibody, Molecular probe
  • DAPI 4,',6-Diamidin-2-phenylindole dihydrochloride, SIGMA
  • 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).
  • Alexa 594 Anti-Rabbit IgG Antibody, Molecular probe
  • Alexa 488 Anti-Mouse IgG Antibody, Molecular probe
  • DAPI 4',6-Diamidin-2-phenylindole dihydrochloride, SIGMA
  • 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 ⁇ 4-l>, respectively, and treated with paraformaldehyde to fix the cells, followed by treating with triton X-100 to improve cell permeability.
  • FSPl Troponin-Linked Fibroblast Surface Protein Clone IBlO, SIGMA
  • 1% skim milk in PBS 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.
  • 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
  • CHEMICON Anti-Rabbit Polyclonal Antibody
  • Alexa 594 Anti-Rabbit IgG Antibody, Molecular probe
  • Alexa 488 Anti-Mouse IgG Antibody, Molecular probe
  • DAPI 4',6-Diamidin-2-phenylindole dihydrochloride, SIGMA
  • 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.
  • 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.
  • 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 4. 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 ⁇ i.
  • the reaction solution was incubated at 65 ° C for 5 minutes, followed by keeping at 0 ° C for a minute.
  • 10 ⁇ of a cDNA synthetic mixture (10*RT buffer, 25 mM MgCl 2 , 0.1 M DTT, RNase OUT, Superscript m 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 ⁇ i 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 0 C for 1 minute.
  • GAPDH was used as a control
  • forward and reverse primer pairs used in the PCR for the amplification of each marker protein were as follows:
  • Pax3, Snail, Slug, nestin and vimentin are expressed at the mRNA level in the dermis-derived cells of the present invention.
  • vimentin was abundantly expressed in the dermis-derived cells of the present invention.
  • the soft tissue filler composition comprising an autologous dermis-derived cell culture material and hyaluronic acid as an effective ingredient according to the present invention shows the complementary action of both hyaluronic acid responsible for a rapid onset of therapeutic effect and the autologous dermis-derived cell culture material responsible for a long-term maintenance thereof without causing any immune response and side-effect. Accordingly, it can be effectively used for smoothing and removing wrinkles and scars as well as improving skin tone and resilience.

Abstract

The present invention relates to a soft tissue filler composition for injection. In particular, the present invention relates to a soft tissue filler composition for injection comprising an autologous dermis-derived cell culture material obtained by separating a dermal biopsy from the patient's own skin and proliferating the same via in vitro serum-free cultivation and hyaluronic acid.

Description

SOFT TISSUE FILLER COMPOSITION COMPRISING AUTOLOGOUS DERMIS-DERIVED CELL CULTURE PRODUCT
AND HYALURONIC ACID
Field of the Invention
The present invention relates to a soft tissue filler composition for injection comprising an autologous dermis-derived cell culture material and hyaluronic acid which is useful for alleviating and removing wrinkles and scars.
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. Approximately twenty years ago, a method of employing bovine collagen for the treatment of wrinkles and scars was developed. However, bovine collagen induced allergic and heterologous rejection reactions to the patients being treated. Further, once implanted, the collagen is rapidly absorbed back into the tissue within a short time and its therapeutic effects can be maintained only for a few months. To solve these problems, a method of preparing collagen by using a patient's own skin and injecting the same to the patient has been developed. However, said method requires a large quantity of the patient's own skin for preparing an effective amount of collagen. Further, the problem of rapid absorption back into the tissue within a short amount of time still remained unsolved. Further, cross-linked collagen proteins with different sizes were used as a soft tissue filler for the last few years. However, the duration of their therapeutic effects was limited to only about 6 months and they caused allergic reactions. To overcome such a immune response of the cross-linked collagen proteins, Autologen prepared by extracting collagen from human placenta was developed. However, it also showed short-term therapeutic effects. Fibrel, the source of which is porcine collagen, was approved by FDA for the treatment of depressed scars and rhytides in 1990. However, it was impossible to expect prolonged therapeutic effects and it tended to cause a side-effect of forming particles at the injection area. Further, Artecoll is a complex of artificially synthesized microspores and bovine collagen. 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.
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 fibrocytes. 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. 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. Further, non-animal stabilized hyaluronic acid available under the trade name Restyland™ (Q-Med Scandinavia Inc., USA) was approved by FDA on April, 2002 as an injection for smoothening wrinkles and augmenting the lips. Hyaluronic acid is a naturally-occurring linear polysaccharide with repeating disaccharide units composed of gluconic acid and N-acetyl-glucosamine, It functions to provide nutrients to the tissue and cells simultaneously with offering liquid environment useful for tissue metabolism and makes the skin resilient and smooth. When the hyaluronic acid is employed as soft tissue filler, it may be advantageous in that allergic reactions and side-effects may be avoided since it is a natural substance. However, the hyaluronic acid may be disadvantageous since the duration of therapeutic effect is too short.
Since the prior art autologous skin cell injections take 3 to 9 months for showing therapeutic effects per unit dose, it is impossible to expect a rapid onset of therapeutic effects. Further, when removing wrinkles or augmenting the lips by using hyaluronic acid, its therapeutic effects are immediately achieved. However, there are still problems in that they are maintained only for about 6 months. Therefore, the present inventors have endeavored to develop a soft tissue filler capable of showing immediate therapeutic effects for alleviating and removing wrinkles and scars simultaneously with maintaining these therapeutic effects for a long time. The present inventors found that a soft tissue filler composition comprising an autologous dermis-derived cell culture material, which is prepared by separating from the patient's own skin and proliferating via in vitro serum-free cultivation, and hyaluronic acid as an effective ingredient shows continuous natural and good therapeutic effects. Thus, such a composition can be effectively used for removing wrinkles and scars.
Disclosure
Technical Problem
Accordingly, the primary object of the present invention is to provide a soft tissue filler composition showing immediate and continuous remarkable therapeutic effects for treating skin damage due to dermal defects.
Technical Solution
In accordance with one aspect of the present invention, there is provided a soft tissue filler composition for injection, which comprises an autologous dermis-derived cell culture material and hyaluronic acid as an effective ingredient, wherein the autologous dermis-derived cell culture material is prepared by separating from the patient's own skin and proliferating via in vitro serum- free cultivation.
The autologous dermis-derived cell culture material of the present invention may be obtained by separating a dermal biopsy from the patient's own skin and proliferating the same via in vitro serum-free cultivation. Such material is a complex of dermis-derived cells containing dermal fibroblast stem cells, dermal fibroblast transit amplifying cells and dermal fibroblasts. The autologous dermis-derived cell culture material of the present invention may further comprise collagen obtained together with said dermis-derived cells during the in vitro serum-free cultivation.
In a preferred embodiment of a soft tissue filler composition according to the present invention, the dermal fibroblast stem cells, dermal fibroblast transit amplifying cells, dermal fibroblasts and collagen in the filler composition are derived from a small amount of the patient's own skin (1 to 30 mm2). In particular, an autologous dermal biopsy isolated from the patient's own skin is digested and separated into single cells. Then, thus separated single dermal cells are subjected to in vitro cultivation and proliferation in an in vitro serum- free culture medium, thereby obtaining an autologous dermis-derived cell culture material containing dermal fibroblast stem cells, dermal fibroblast transit amplifying cells, dermal fibroblasts and collagen as a complex filler.
In a preferred embodiment, the autologous dermis-derived cell culture material in the soft tissue filler composition of the present invention may be prepared by the following steps:
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; and 3) centrifuging the autologous dermis-derived cell culture material to separate an autologous dermis-derived cell pellet.
The autologous dermis-derived cell material obtained according to the method of the present invention is composed of dermis-derived cells containing dermal fibroblast stem cells, dermal fibroblast transit amplifying cells and dermal fibroblasts proliferated by in vitro serum-free cultivation, as well as collagen secreted therefrom. The total number of cells as an effective ingredient in the cell culture material is preferably in the range from I x IO7 to 8x 107 cells/m£, more preferably I x IO7 to 5χ lO7 cellsM, while the content of collagen therein is preferably in the range from 10 to 100 mg/m£, more preferably 20 to 60 mg/m#.
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 mnf 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/ml
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/mt. 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 autologous dermis-derived cell culture material thus prepared contains dermal fibroblast stem cells, dermal fibroblast transit amplifying cells, dermal fibroblasts and collagen as an effective ingredient, wherein the mixed ratio of the dermal fibroblast stem cells, dermal fibroblast transit amplifying cells and dermal fibroblasts is 5 to 20% : 20 to 50% : 30 to 70%, while the content of collagen is in the range from 10 to 100 mg/m#, preferably 20 to 60 mg/m-£.
Hyaluronic acid useful in the present invention may include natural hyaluronic acid isolated from human skin or an animal source and modified hyaluronic acid produced by fermenting genetically engineered microorganisms, e.g., a hyaluronic acid product commercially produced by Shandong Freda Biochem Co., Ltd. Hyaluronic acid is a glycosaminoglycan heteropolysaccharide distributed widely throughout connective, epithelial and neural tissues. It is one of the chief components of the extracellular matrix between epidermal and dermal layers. It is more effective to inject hyaluronic acid having a higher molecular weight for restoring a depressed skin area. However, since hyaluronic acid having a molecular weight of 5,000,000 daltons or more has extremely strong viscosity to use as an injection, it is preferable to employ hyaluronic acid having a molecular weight ranging from 1,000,000 to 5,000,000 daltons. The soft tissue filler composition of the present invention contains hyaluronic acid at a concentration ranging from 2 to 20 wg/mH, preferably 5 to 15 mg/ml
The soft tissue filler composition for removing wrinkles and scars according to the present invention may be prepared by obtaining the autologous dermis-derived cell culture material containing dermal fibroblast stem cells, dermal fibroblast transit amplifying cells, dermal fibroblasts and collagen through the in vitro serum-free cultivation of the patient's own dermal biopsy and mixing it with 1 to 4 ml of hyaluronic acid at a concentration ranging from 2.0 to 20 mg/mβ so as to adjust the final cell concentration to Ix IO7 to 8* 107 cells/ml
Since hyaluronic acid is a natural substance, it is safe and does not cause any allergic reaction or side-effect. Thus, it can be effectively used as an epidermal layer filling material. However, its therapeutic effect lasts only for 6 months. On the other hand, the removal of wrinkles using only autologous dermis-derived cells can safely improve facial wrinkles or depressed scars through the prolonged therapeutic effect, thereby highly satisfying a patient being treated. However, there is a problem in that the onset of such a therapeutic effect is too late. Specifically, the present invention is characterized by mutually complementing the problems caused by the single use of the autologous dermis-derived cells or hyaluronic acid alone through the combination thereof, thereby achieving the purpose of the immediate onset and prolonged maintenance of therapeutic effect.
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.
Since the soft tissue filler composition for injection of the present invention comprises the autologous dermis-derived cell culture material originated from the patient's own skin as an effective ingredient, there is no risk of causing any rejection reaction or side-effect. Further, it can keep up its prolonged therapeutic effect for several years. Also, the injection of the present invention can bring an immediate onset of therapeutic effect due to hyaluronic acid among the dose unit, while the short-term therapeutic effect of hyaluronic acid is complemented by the autologous dermis-derived cells showing therapeutic effects only after 3 to 9 months have passed from the onset of the treatment. This causes the therapeutic effects of the injection to last for a long time. Accordingly, the injection of the present invention has advantages of immediately exerting remarkable filling and restoring effects after application to a target site as well as continuously maintaining such a therapeutic effect for a long time.
Furthermore, since hyaluronic acid used in the injection of the present invention provides a favorable environment for the growth of autologous dermis-derived cells included therein, it can be expected to further produce collagen as these cells grow continuously after the injection. That is, since the injection of the present invention produces additional collagen at the injection site after the treatment besides the collagen originally contained therein, it shows excellent therapeutic effects of smoothening and removing the wrinkles and scars. Further, it is capable of improving the local environment of the skin, which can be effectively used for restoring natural and more youthful appearance in case of applying to the face.
The injection of the present invention exhibits better and more natural therapeutic effects and can maintain the same for a long time compared to injections of bovine collagen, autologous collagen and Botox.
Accordingly, the present invention further comprises a method of alleviating or removing wrinkles or scars by injecting the injection of the present invention to the area having skin damage due to dermal defects, e.g., wrinkles or scars. The method can be effectively used for alleviating or removing wrinkles on the face and neck, stretch marks from pregnancy or wrinkles on the wrist and augmenting the lips.
Advantageous Effect
The soft tissue filler composition comprising an autologous dermis- derived cell culture material and hyaluronic acid as an effective ingredient according to the present invention shows the complementary action of both hyaluronic acid responsible for a rapid onset of therapeutic effect and the autologous dermis-derived cell culture material responsible for a long-term maintenance thereof without causing any immune response and side-effect. Accordingly, it can be effectively used for smoothening and removing the wrinkles and scars as well as improving the skin tone and resilience.
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 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. 2 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)5 wherein nestin is detected as a red spot at 594 nm, fibronectin is detected as a green spot at 488 run and cell nuclei is detected as a blue spot by DAPI staining;
Fig. 3 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. 4 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. 5 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 mnf 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 mi 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 mi 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 rpm/min for 5 minutes to remove a supernatant, thereby recovering only a cell pellet.
To the cell pellet obtained above was added 1 mi of a dermal cell culture medium (Cascade, Cat. No. 106, supplemented with 1 ng/mi 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 2χ 107 to 6x 107 cells/mβ 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 mi 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 mi 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 AfB, 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 (ft ft?) , Lai Xinan (^W ^Q ,
Wang Zhengguo ff lE H) , Wang LiIi (3E BR BB) , 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 (age ranging from 18 to 60) who are 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 11 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 ml of the soft tissue filler composition prepared in Example <1-1> was filled into a 3 m£ 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° . 15 or 20 injection points were taken at the wrinkled area, while 0.05 to 0.1 mi of the filler composition was injected into one point. 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. Within 24 hours after the treatment, light swelling or tingling at the injection area may have occurred. However, there was no local or systemic symptom in the entire body. 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:
O: wrinkles are eliminated at the injection area and the skin is completely restored. Δ: wrinkles are mitigated at the injection area and more clearly improved than before the treatment. x : there is no meaningful change between before and after the treatment in terms of wrinkle improvement. [Table 1]
As illustrated in Table 1, the therapeutic effect of hyaluronic acid on the wrinkle improvement was immediately shown in the patient treated with the injection comprising the autologous dermis-derived cell culture material and hyaluronic acid as an effective ingredient according to the present invention. At the same time, the dermal fibroblast transit-amplifying cells in the autologous dermis-derived cell culture material were gradually matured and converted into the dermal fibroblasts with producing autologous collagen in the course of time, which makes the thickness and density of the dermal layer to be increased, wrinkles and depressed scars to be improved, and the skin tone and resilience to be restored. Said therapeutic effects were maintained for a long time. During treatment, one of the patients being treated had an unpleasant feeling due to the outbreak of reddish acne at the injection area after the third injection. However, the acne was improved without any further treatment 2 days after the outbreak. Further, abnormal symptoms were not reproduced during the treatment. This confirms the safety of the composition according to the present invention.
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 3> 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 mπf. 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/ml of hEGF, 3 ng/iM 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 370C 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, 1, 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 4> Immunological comparison of dermis-derived cells with adipose tissue-derived cells
<4-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 3. 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 3. 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-IOO 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 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. 2, nestin and fibronectin 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).
<4-2> 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 <4-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. 3, 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).
<4-3> 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 <4-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 IBlO, 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. 4, 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 5> 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 4. 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 μi. The reaction solution was incubated at 65 °C for 5 minutes, followed by keeping at 0°C for a minute. After 10 ≠ of a cDNA synthetic mixture (10*RT buffer, 25 mM MgCl2, 0.1 M DTT, RNase OUT, Superscript m 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 μi 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 720C 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 pax3-2 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. 5, 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 comprising an autologous dermis-derived cell culture material and hyaluronic acid as an effective ingredient according to the present invention shows the complementary action of both hyaluronic acid responsible for a rapid onset of therapeutic effect and the autologous dermis-derived cell culture material responsible for a long-term maintenance thereof without causing any immune response and side-effect. Accordingly, it can be effectively used for smoothing and removing wrinkles and scars as well as improving skin tone and resilience.

Claims

What is claimed is:
1. A soft tissue filler composition for alleviating or treating skin damage due to dermal defects, comprising:
(a) an autologous dermis-derived cell culture material containing dermal fibroblast stem cells, dermal fibroblast transit-amplifying cells and dermal fibroblasts obtained by separating a dermal biopsy from a patient's own skin and proliferating the same via in vitro serum-free cultivation, and collagen secreted therefrom; and
(b) hyaluronic acid.
2. The soft tissue filler composition according to Claim 1, wherein the concentration of the autologous dermis-derived cells in the cell culture material is in the range from 1 x 107 to 8χ 107 cells/ml
3. The soft tissue filler composition according to Claim 2, wherein the ratio of dermal fibroblast stem cells, dermal fibroblast transit-amplifying cells and dermal fibroblasts in the autologous dermis-derived cell culture material is 5 to 20% : 20 to 50% : 30 to 70%.
4. The soft tissue filler composition according to Claim 1, wherein the content of collagen in the autologous dermis-derived cell culture material is in the range from 10 to 100 mg/ml
5. The soft tissue filler composition according to Claim 1, wherein the autologous dermis-derived cell culture material is prepared by the following steps:
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; and
3) centrifuging the autologous dermis-derived cell culture material to separate an autologous dermis-derived cell pellet.
6. The soft tissue filler composition according to Claim 5, wherein the medium for in vitro serum-free cultivation in Step 2) contains a growth factor and an activation factor.
7. The soft tissue filler composition according to Claim 6, 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.
8. The soft tissue filler composition according to Claim 7, wherein the content of the growth factor is in the range from 0.1 to 1 ng/m#.
9. The soft tissue filler composition according to Claim 6, 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.
10. The soft tissue filler composition according to Claim 9, wherein the content of the activation factor is in the range from 0.1 to 1%.
11. The soft tissue filler composition according to Claim 1 , wherein the in vitro serum-free cultivation is conducted for 6 to 10 weeks.
12. The soft tissue filler composition according to Claim 1, wherein the hyaluronic acid is included at a concentration ranging from 2 to 20 mg/ml.
13. The soft tissue filler composition according to Claim 1, wherein the hyaluronic acid is a natural hyaluronic acid isolated from an animal source or a modified hyaluronic acid produced by fermenting genetically engineered microorganisms.
14. The soft tissue filler composition according to Claim 1, wherein the hyaluronic acid has a molecular weight ranging from 1,000,000 to 5,000,000 daltons.
15. A soft tissue filling injection for alleviating or treating skin damage due to dermal defects which is formulated with the composition according to Claim 1.
16. The soft tissue filling injection according to Claim 15, wherein the alleviation or treatment of skin damage due to dermal defects is intended for reducing wrinkles, removing scars or augmenting the lips.
17. The soft tissue filling injection according to Claim 16, wherein the wrinkles include wrinkles on the face and neck, stretch marks from pregnancy and wrinkles on the back of the hand.
18. The soft tissue filling injection according to any one of Claims 15 to 17, further comprising pharmaceutically acceptable excipients and/or additional agents.
19. The soft tissue filling injection according to Claim 18, wherein the additional agent is selected from the group consisting of a carrier, a diluent, an excipient, a thickening agent, an emulsifying agent, a preservative and a mixture thereof.
EP07747120A 2006-06-26 2007-06-26 Soft tissue filler composition comprising autologous dermis-derived cell culture product and hyaluronic acid Withdrawn EP2049130A4 (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9610430B2 (en) 2006-09-11 2017-04-04 Renovacare Sciences Corp. Cell spraying device, method and sprayed cell suspension
US10376658B2 (en) 2011-04-27 2019-08-13 Renovacare Sciences Corp. Device for cell spraying
US11040363B2 (en) 2016-06-14 2021-06-22 Renovacare Sciences Corp. Modular device for cell spraying

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101421049B1 (en) * 2008-02-29 2014-07-21 (주)아모레퍼시픽 Cosmetic composition for lip-plumping and wrinkle-improvement of lips
PL3357519T3 (en) * 2008-07-02 2019-09-30 Allergan, Inc. Compositions for soft tissue filling and regeneration
US9173975B2 (en) 2009-04-24 2015-11-03 Ingeneron, Inc. Reparative cell delivery via hyaluronic acid vehicles
KR101145394B1 (en) * 2009-07-29 2012-05-15 주식회사 바이오랜드 Compositions for promoting stemness and proliferation of skin cells comprising Oligo-hyaluronic acid
JP6023049B2 (en) * 2010-06-30 2016-11-09 チェン，ジンシー Method for producing aggregate of skin tissue cells and use thereof
WO2014133339A1 (en) * 2013-02-28 2014-09-04 (주)아모레퍼시픽 Composition for maintaining efficacy of filler
KR102459312B1 (en) * 2020-09-15 2022-10-26 주식회사 유나이티드엑티브 Fermentation method for the preparation of hyaluronic acid with hydrophobic properties
KR102358306B1 (en) 2020-10-08 2022-02-07 김예주 Injectable composition for neck wrinkles
CN113456574A (en) * 2021-07-09 2021-10-01 上海鄄飞健康管理咨询有限公司 Nutrient solution with neck anti-aging function and use method thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000037124A1 (en) * 1998-12-21 2000-06-29 Fidia Advanced Biopolymers, S.R.L. Injectable hyaluronic acid derivative with pharmaceuticals/cells
US20020197241A1 (en) * 1998-05-22 2002-12-26 Isolagen Technologies, Inc. A Delaware Corporation Compositions for regenerating tissue that has deteriorated, and methods for using such compositions
EP1358857A1 (en) * 1995-07-28 2003-11-05 Isolagen International S.A. The use of autologous dermal fibroblasts for the repair of skin and soft tissue defects
WO2006125991A1 (en) * 2005-05-26 2006-11-30 Intercytex Limited Tissue repair using allogenic dermal fibroblasts
WO2007139525A2 (en) * 2006-05-31 2007-12-06 Kemal Tunc Tiryaki A method and a tissue material for use in the treatment of the skin defects and aging

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5965125A (en) * 1995-10-25 1999-10-12 Transkaryotic Therapies, Inc. Hybrid matrix implants and explants
CA2319558A1 (en) * 1998-02-27 1999-09-02 Bioelastics Research, Ltd. Injectable implants for tissue augmentation and restoration
US8394371B2 (en) * 2002-02-11 2013-03-12 Neocutis Sa Compositions for the treatment of skin conditions, disorders or diseases and methods of making and using the same

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1358857A1 (en) * 1995-07-28 2003-11-05 Isolagen International S.A. The use of autologous dermal fibroblasts for the repair of skin and soft tissue defects
US20020197241A1 (en) * 1998-05-22 2002-12-26 Isolagen Technologies, Inc. A Delaware Corporation Compositions for regenerating tissue that has deteriorated, and methods for using such compositions
WO2000037124A1 (en) * 1998-12-21 2000-06-29 Fidia Advanced Biopolymers, S.R.L. Injectable hyaluronic acid derivative with pharmaceuticals/cells
WO2006125991A1 (en) * 2005-05-26 2006-11-30 Intercytex Limited Tissue repair using allogenic dermal fibroblasts
WO2007139525A2 (en) * 2006-05-31 2007-12-06 Kemal Tunc Tiryaki A method and a tissue material for use in the treatment of the skin defects and aging

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
"IBMS-ECTS 2005 Abstracts", BONE, PERGAMON PRESS., OXFORD, GB, vol. 36, 1 January 2005 (2005-01-01), pages S103-S479, XP025396852, ISSN: 8756-3282 [retrieved on 2005-01-01] *
See also references of WO2008002063A1 *
YOON EUL-SIK ET AL: "Advantages of the Presence of Living Dermal Fibroblasts Within Restylane for Soft Tissue Augmentation" ANNALS OF PLASTIC SURGERY, LITTLE, BROWN AND CO, US, vol. 51, no. 6, 1 December 2003 (2003-12-01), pages 587-592, XP008093679 ISSN: 0148-7043 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9610430B2 (en) 2006-09-11 2017-04-04 Renovacare Sciences Corp. Cell spraying device, method and sprayed cell suspension
US10376658B2 (en) 2011-04-27 2019-08-13 Renovacare Sciences Corp. Device for cell spraying
US11135380B2 (en) 2011-04-27 2021-10-05 Renovacare Sciences Corp. Device for cell spraying
US11040363B2 (en) 2016-06-14 2021-06-22 Renovacare Sciences Corp. Modular device for cell spraying

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