WO2008002064A1 - Composition de charge de tissus mous pour injection et son procédé de préparation - Google Patents

Composition de charge de tissus mous pour injection et son procédé de préparation Download PDF

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Publication number
WO2008002064A1
WO2008002064A1 PCT/KR2007/003100 KR2007003100W WO2008002064A1 WO 2008002064 A1 WO2008002064 A1 WO 2008002064A1 KR 2007003100 W KR2007003100 W KR 2007003100W WO 2008002064 A1 WO2008002064 A1 WO 2008002064A1
Authority
WO
WIPO (PCT)
Prior art keywords
dermal
cells
injection
autologous
filler composition
Prior art date
Application number
PCT/KR2007/003100
Other languages
English (en)
Inventor
Bin Han
Jay Do Choi
Min Jung Nam
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 WO2008002064A1 publication Critical patent/WO2008002064A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0656—Adult fibroblasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides

Definitions

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

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Veterinary Medicine (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Biotechnology (AREA)
  • Dermatology (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Developmental Biology & Embryology (AREA)
  • Rheumatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Virology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé de préparation d'une composition de charge de tissus mous pour injection, comprenant les étapes suivantes: 1) la digestion d'un tissu dermique autologue isolé de la peau du patient et la séparation en cellules isolées; 2) la culture et la prolifération des cellules dermiques isolées en culture in vitro en milieu exempt de sérum pour obtenir un matériau autologue de culture cellulaire dérivé du derme contenant des cellules souches de fibroblaste dermique, des cellules amplificatrices transitoires de fibroblaste dermique, et des fibroblastes et du collagène dermique; 3) la centrifugation du matériau autologue de culture cellulaire dérivée du derme pour séparer une pastille cellulaire autologue dérivée du derme; et 4) la suspension de la pastille cellulaire autologue dérivée du derme dans une injection de glucose ou une injection aléatoire pour obtenir une suspension pour injection. L'invention concerne également une composition de charge de tissus mous pour injection préparée par le procédé selon l'invention.
PCT/KR2007/003100 2006-06-26 2007-06-26 Composition de charge de tissus mous pour injection et son procédé de préparation WO2008002064A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2006-0057697 2006-06-26
KR1020060057697A KR20070122316A (ko) 2006-06-26 2006-06-26 주사용 인체 연조직 충전제 및 이의 제조 방법

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RU (1) RU2396084C1 (fr)
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Cited By (32)

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WO2011140323A1 (fr) * 2010-05-07 2011-11-10 Fibrocell Science, Inc. Formulations d'unités de dosage de fibroblastes dermiques autologues
WO2012051505A1 (fr) * 2010-10-14 2012-04-19 Fibrocell Science, Inc. Traitement de cordes vocales par une formulation de fibroblastes dermiques autologues
US9987473B2 (en) 2009-12-18 2018-06-05 Srgi Holdings, Llc Skin treatment device and methods
US10076354B2 (en) 2010-12-17 2018-09-18 Srgi Holdings, Llc Pixel array medical devices and methods
US10219827B2 (en) 2010-12-17 2019-03-05 Srgi Holdings, Llc Pixel array medical devices and methods
US10314640B2 (en) 2010-12-17 2019-06-11 Srgi Holdings, Llc Pixel array medical devices and methods
US10335190B2 (en) 2013-12-06 2019-07-02 Srgi Holdings, Llc Pixel array medical systems, devices and methods
US10368904B2 (en) 2013-12-06 2019-08-06 Srgi Holdings, Llc Pixel array medical systems, devices and methods
CN110192989A (zh) * 2019-05-07 2019-09-03 深圳欧珈再生医学抗衰生物工程有限公司 用于促进皮肤干细胞和胶原母细胞再生组合物及制备方法
CN110269868A (zh) * 2019-06-12 2019-09-24 江苏艾尔康生物医药科技有限公司 一种含有自体真皮成纤维细胞透明质酸凝胶剂的构建方法
US10517635B2 (en) 2013-12-06 2019-12-31 Srgi Holdings Llc Pixel array medical systems, devices and methods
US10561737B2 (en) 2014-01-03 2020-02-18 Hoffmann-La Roche Inc. Bispecific anti-hapten/anti-blood brain barrier receptor antibodies, complexes thereof and their use as blood brain barrier shuttles
US10661063B2 (en) 2010-12-17 2020-05-26 Srgi Holdings, Llc Systems, devices and methods for fractional resection, fractional skin grafting, fractional scar reduction and fractional tattoo removal
US10695546B2 (en) 2010-12-17 2020-06-30 Srgi Holdings, Llc Systems, devices and methods for fractional resection, fractional skin grafting, fractional scar reduction and fractional tattoo removal
US10702684B2 (en) 2010-12-17 2020-07-07 Srgi Holdings, Llc Systems, devices and methods for fractional resection, fractional skin grafting, fractional scar reduction and fractional tattoo removal
US10736653B2 (en) 2013-12-06 2020-08-11 Srgi Holdings, Llc Pixel array medical systems, devices and methods
US10772658B2 (en) 2010-12-17 2020-09-15 Srgi Holdings, Llc Pixel array medical systems, devices and methods
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US11051844B2 (en) 2010-12-17 2021-07-06 Srgi Holdings, Llc Pixel array medical devices and methods
CN113181219A (zh) * 2021-03-04 2021-07-30 纳美细胞科技有限公司 用于除皱的细胞制剂及其制备方法
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CN114344454A (zh) * 2022-01-21 2022-04-15 郑州市金水区蕾娜斯医疗美容门诊部 自体脂肪胶原蛋白注射剂的制备方法
US11490952B2 (en) 2015-08-31 2022-11-08 Srgi Holdings, Llc Pixel array medical devices and methods
US11564706B2 (en) 2019-10-28 2023-01-31 Srgi Holdings, Llc Pixel array medical systems, devices and methods
US11673968B2 (en) 2014-06-26 2023-06-13 Hoffmann-La Roche Inc. Anti-BRDU antibodies and methods of use
CN116474172A (zh) * 2023-04-17 2023-07-25 广州市麦施缔医疗科技有限公司 表皮干细胞与脂肪混合注射方法和隆胸及***再生方法
US11751904B2 (en) 2015-08-31 2023-09-12 Srgi Holdings, Llc Pixel array medical systems, devices and methods
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US11980389B2 (en) 2015-08-31 2024-05-14 Srgi Holdings Llc Handed spiral slotted scalpet array
US12023378B2 (en) 2012-07-04 2024-07-02 Hoffmann-La Roche Inc. Covalently linked antigen-antibody conjugates

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RU2749801C1 (ru) * 2020-03-06 2021-06-17 Галина Мироновна Могильная Способ восстановления утраченного объема дермы в эксперименте на крысах

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US11090473B2 (en) 2009-12-18 2021-08-17 Srgi Holdings, Llc Skin treatment device
US10773064B2 (en) 2009-12-18 2020-09-15 Srgi Holdings, Llc Skin treatment device and methods
US11554142B2 (en) 2010-05-07 2023-01-17 Castle Creek Biosciences, Llc Dosage unit formulations of autologous dermal fibroblasts
AU2011248067B2 (en) * 2010-05-07 2015-03-12 Castle Creek Biosciences, Llc Dosage unit formulations of autologous dermal fibroblasts
CN107812014A (zh) * 2010-05-07 2018-03-20 法布罗赛尔科技公司 自体真皮成纤维细胞的剂量单位调配物
US8529883B2 (en) 2010-05-07 2013-09-10 Fibrocell Technologies, Inc. Dosage unit formulations of autologous dermal fibroblasts
CN102905711A (zh) * 2010-05-07 2013-01-30 法布罗赛尔科技公司 自体真皮成纤维细胞的剂量单位调配物
WO2011140323A1 (fr) * 2010-05-07 2011-11-10 Fibrocell Science, Inc. Formulations d'unités de dosage de fibroblastes dermiques autologues
US8765121B2 (en) 2010-10-14 2014-07-01 Fibrocell Technologies, Inc. Treatment of vocal cords with autologous dermal fibroblast formulation
US9415075B2 (en) 2010-10-14 2016-08-16 Fibrocell Technologies, Inc. Treatment of vocal cords with autologous dermal fibroblast formulation
WO2012051505A1 (fr) * 2010-10-14 2012-04-19 Fibrocell Science, Inc. Traitement de cordes vocales par une formulation de fibroblastes dermiques autologues
US10098914B2 (en) 2010-10-14 2018-10-16 Fibrocell Technologies, Inc. Treatment of vocal cords with autologous dermal fibroblast formulation
US10881695B2 (en) 2010-10-14 2021-01-05 Fibrocell Technologies, Inc. Treatment of vocal cords with autologous dermal fibroblast formulation
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US10076354B2 (en) 2010-12-17 2018-09-18 Srgi Holdings, Llc Pixel array medical devices and methods
US11278309B2 (en) 2010-12-17 2022-03-22 Srgi Holdings, Llc Pixel array medical systems, devices and methods
US10661063B2 (en) 2010-12-17 2020-05-26 Srgi Holdings, Llc Systems, devices and methods for fractional resection, fractional skin grafting, fractional scar reduction and fractional tattoo removal
US10695546B2 (en) 2010-12-17 2020-06-30 Srgi Holdings, Llc Systems, devices and methods for fractional resection, fractional skin grafting, fractional scar reduction and fractional tattoo removal
US10702684B2 (en) 2010-12-17 2020-07-07 Srgi Holdings, Llc Systems, devices and methods for fractional resection, fractional skin grafting, fractional scar reduction and fractional tattoo removal
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US10967162B2 (en) 2010-12-17 2021-04-06 Srgi Holdings, Llc Systems, devices and methods for fractional resection, fractional skin grafting, fractional scar reduction and fractional tattoo removal
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US10080581B2 (en) 2010-12-17 2018-09-25 Srgi Holding Llc Pixel array medical devices and methods
US12023378B2 (en) 2012-07-04 2024-07-02 Hoffmann-La Roche Inc. Covalently linked antigen-antibody conjugates
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