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WO1993004164A1 - Milieu conditionne derive des keratinocytes permettant de produire des facteurs de croissance - Google Patents

Milieu conditionne derive des keratinocytes permettant de produire des facteurs de croissance Download PDF

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Publication number
WO1993004164A1
WO1993004164A1 PCT/US1991/006161 US9106161W WO9304164A1 WO 1993004164 A1 WO1993004164 A1 WO 1993004164A1 US 9106161 W US9106161 W US 9106161W WO 9304164 A1 WO9304164 A1 WO 9304164A1
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WO
WIPO (PCT)
Prior art keywords
kdcmf
conditioned medium
factors
epithelial cells
medium
Prior art date
Application number
PCT/US1991/006161
Other languages
English (en)
Inventor
Gary D. Shipley
Paul W. Cook
Original Assignee
THE STATE OF OREGON acting by and through THE STATE OF BOARD OF HIGHER EDUCATION on behalf of THE OREGON HEALTH SCIENCES UNIVERSITY
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 THE STATE OF OREGON acting by and through THE STATE OF BOARD OF HIGHER EDUCATION on behalf of THE OREGON HEALTH SCIENCES UNIVERSITY filed Critical THE STATE OF OREGON acting by and through THE STATE OF BOARD OF HIGHER EDUCATION on behalf of THE OREGON HEALTH SCIENCES UNIVERSITY
Publication of WO1993004164A1 publication Critical patent/WO1993004164A1/fr

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Classifications

    • 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
    • A61K38/18—Growth factors; Growth regulators
    • 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

Definitions

  • This invention is in the general field of wound healing. More specifically it relates to a novel method of producing human keratinocyte-derived conditioned medium factors (kdCMF) which promote healing of surface wounds, ulcerations, and other hypoproliferative skin conditions.
  • kdCMF human keratinocyte-derived conditioned medium factors
  • the growth of cells may be regulated by both autocrine and paracrine growth factors.
  • Autocrine growth factors are those secreted by growing cells which factors stimulate or inhibit the proliferation of the same cell.
  • Paracrine growth factors are those that act on neighboring cells.
  • human keratinocytes produce bFGF (Halaban et al., J Cell Biol (1988) 107:1611-1619), PDGF (Ansel et al., J Invest De ⁇ natol (1990) 94.:101s-107s) and interleukins (Blanton et al., Proc Natl Acad Sci USA (1989) __.'1273-1277; Kirnbauer et al. , J Immunol (1989) 142.:1922-1928) .
  • the present invention involves the preparation and use of a mixture of growth factors, both autocrine and paracrine, produced by human epithelial cells.
  • This represents a desirable combination of factors capable of effecting wound healing and tissue repair.
  • Such combinations of factors in their native proportion are particularly desirable for therapeutic purposes and can be so used by itself or in a pharmaceutical formulation.
  • This mixture of growth factors is produced by harvesting conditioned medium from cultures of human epithelial cells grown in protein-free medium.
  • the invention relates to a method to produce human keratinocyte-derived conditioned medium factors (kdCMF) , which method comprises culturing human epithelial cells in a protein-free medium to obtain a conditioned medium and recovering the conditioned medium from the culture.
  • kdCMF human keratinocyte-derived conditioned medium factors
  • Another aspect of the invention relates to the use of kdCMF to promote healing of surface wounds, ulcerations and other hypoproliferative skin pathologies in humans, and to pharmaceutical compositions useful for this purpose
  • Still another aspect of the invention relates to the use of kdCMF as a supplement to cell culture medium for the purpose of promoting cell growth and viability of cells other than human keratinocytes.
  • An object of the invention is to provide a novel mixture of growth factors which growth factors include autocrine and paracrine growth factors which growth factors are present with respect to each other in substantially the same relative amounts in which they naturally occur indigenously within a mammal such as a human.
  • An advantage of the present invention is that the mixture of growth factors provides efficacious results when applied to a wound in terms of increasing the rate of wound healing.
  • a feature of the present invention is that the mixture of growth factors is non-toxic in that it includes naturally occurring growth factors in their naturally occurring proportional amounts.
  • kdCMF keratinocyte-conditioned medium factors
  • kdCMF refers to the mixture of autocrine and paracrine factors which are produced by culturing human epithelial cells in a protein-free medium.
  • the medium will contain a mixture of these factors produced by the cells.
  • the kdCMF of the invention is simply the conditioned medium harvested from such cultures.
  • the conditioned medium itself can also be further fractionated (using standard separation protocols based on charge, hydrophobicity, size or ligand affinity) into two or several fractions which retain mixtures of the secreted factors, although in altered ratio.
  • Such crude fractions can also be used in the wound-healing methods and cell proliferation methods of the invention and are another embodiment of kdCMF.
  • the conditioned medium can also be concentrated and/or freed of inorganic and small organic materials by dialysis and/or lyophilization and/or other methodologies.
  • the resulting concentrated form of the conditioned medium also contains these factors, and is thus a more convenient form of kdCMF.
  • the invention involves the purified mixture of growth factors which are obtained from the protein-free medium on which the epithelial cells are allowed to grow.
  • the growth factors are purified but maintained so that the natural ratio of each of the growth factors to each other remains substantially undisturbed. It is believed that by maintaining the particular ratio of each growth factor to the other, it is possible to obtain a mixture of growth factors which is both safe and efficacious with respect to enhancing the rate of wound healing.
  • defined medium refers to medium which contains no crude extracts or common supplements such as pituitary extract, serum proteins, and so forth.
  • Defined medium refers to the conventional understanding as it pertains to growth of cells in a medium containing no undefined additives, as that term is commonly used in the art.
  • protein-free standard medium also lacks exogenous artificial sources of specific growth factors such as EGF, TGF ⁇ , or other polypeptides.
  • EGF EGF
  • TGF ⁇ transforming growth factor
  • complete medium refers to high amino-acid (HAA) modified medium MCDB 153 (Pittelkow and Scott, Mayo Clin Proc (1986) 6_1:771-777, incorporated herein by reference to disclose such a medium) supplemented with 0.2% (v/v) bovine pituitary extract (BPE) , culture grade EGF (10 ng/ml) , insulin (5 ⁇ g/ml) , hydrocortisone (5 x 10 M) , ethanolamine (1 x 10 M) and phosphoethanolamine (1 x 10 M) , gentamicin sulfate ( ⁇ g/ml) or in KBM medium (Clonetics Corp.) with the same supplements.
  • HAA high amino-acid
  • BPE bovine pituitary extract
  • defined medium refers to complete medium without BPE.
  • Protein-free standard medium as illustrated herein as “protein-free standard medium-1” refers to complete medium without BPE, EGF and insulin.
  • the kdCMF can be produced using a variety of protocols which are similar to that described in Example 1 herein, and generally by plating human epithelial cells in complete, defined or protein-free standard medium at varying densities in different cell culture vessels or on different substrates.
  • Alternative protocols utilize other complete, defined or protein-free media to plate the human keratinocytes. Once plated, the medium is changed to protein-free standard medium to facilitate the isolation of kdCMF. While human keratinocytes are preferred, related cell types (other human epithelial cells) which may or may not be immortalized are viable alternatives in the generation of kdCMF.
  • the present invention involves the use of human epithelial cells and more specifically human keratinocytes
  • the present invention is intended to encompass other mixtures of growth factors which can be obtained from the culturing of non-human mammalian epithelial cells and non-human mammalian keratinocytes.
  • certain modifications to the medium upon which those cells are grown should be made in order to allow for the efficient growth of such non- human cells. It may be most desirable to obtain certain non-human cells such as porcine epithelial cells or porcine keratinocytes in that the growth factors and growth factor mixtures produced by such cells might well be readily amenable for human use.
  • the present invention includes mixtures of human and non- human growth factors obtained from the culturing of human and non-human epithelial cells and specifically human and non-human keratinocytes, as well as pharmaceutical formulations containing such mixtures of growth factors and methods of using such mixtures and formulations in order to promote wound healing.
  • the human skin consists of a vascularized dermis that is separated by a basement membrane from the avascular epidermis.
  • the epidermis is composed of several topologically organized compartments including a proliferative basal layer and post-mitotic suprabasal layers which differentiate and form the keratinized outer layer of the skin. It is generally regarded that proliferating keratinocytes within the basal layer of the epidermis must rely on the vascularized dermal layers for nutritive support.
  • the wound-healing process is clearly a complicated sequence of events which may involve neovascularization, synthesis of extracellular matrix components and stimulation of cell migration and proliferation.
  • the exact identities and quantities of factors necessary for expedient healing of surface wounds in humans are not known, and all of the factors which regulate the processes of wound-healing are not completely understood.
  • the application of single growth factors (FGFs, EGF, TGF ⁇ , PDGFs, TGFjSs) to promote the healing of wounds and ulcerations is of less benefit than the addition of preparations which contain factors that more closely mimic the array and concentration of factors produced by epidermal cells at the site of injury.
  • kdCMF is a complete mixture of keratinocyte-derived factors capable of expediting the wound-healing process in a manner similar to that mediated by epidermal cells at the site of injury. kdCMF also offers advantages over the use of keratinocytes per se.
  • kdCMF The conditions which may benefit from the application of kdCMF include, but are not limited to: epidermal ulcerations (decubitus ulcers, ischemic ulcers, infarctive ulcers, vascular ulcers, and hemoreologic ulcers) , surface wounds (thermal and chemical induced burns, abrasions, lacerations, incisions, skin graft donor sites, and skin graft recipient sites) , lupus erythematosus, corticosteroid-induced atrophy, pemphigus, pemphigoid, androgenetic alopecia and alopecia areata.
  • epidermal ulcerations decubitus ulcers, ischemic ulcers, infarctive ulcers, vascular ulcers, and hemoreologic ulcers
  • surface wounds thermal and chemical induced burns, abrasions, lacerations, incisions, skin graft donor sites, and skin graft recipient sites
  • the active ingredient is formulated into suitable topical compositions, including salves, creams, lotions, solutions, and the like. Additional excipients and palliative factors may also be added.
  • suitable topical compositions are well known in the art and may be found, for example, in Remington's Pharmaceutical Sciences. latest edition, Mack Publishing Company, Easton, PA.
  • compositions useful in wound healing may also include autologous cells (normal human keratinocytes and/or fibroblasts) or other purified growth factors (TGFC-, TGF/8, FGFs, HB-EGF, EGF, AR, PDGFs, epithelins) . Simultaneous or additional administration of these cells and/or factors will be of benefit in mediating the wound- healing process.
  • kdCMF with or without the above-described additives with bandages, or with solid matrices or supports (collagen, collagen- glycosaminoglycan) will be of value as effective methods of delivering kdCMF to lesions and wounds.
  • the kdCMF of the invention may be used as a supplement in culturing various cells which can benefit from this array of growth factors.
  • Suitable cell types which can be benefited in this way include various epithelial cultures, such as those derived from the skin trachea, bronchia, urogenital tract and mammary epithelium.
  • epithelial cultures such as those derived from the skin trachea, bronchia, urogenital tract and mammary epithelium.
  • the following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make the growth factor mixtures of the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, time, etc.), but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in degrees centigrade and pressure is at or near atmospheric.
  • Keratinocytes are plated in complete or defined medium at a density of 1-5 x 10 3 cells/cm2 in tissue culture dishes or other suitable vessels. After 1-3 days of incubation, the cells are washed 3 times with Hepes buffered normal saline and the medium replaced with protein-free standard medium. After 24 hours of additional incubation, the medium is discarded and replaced with fresh protein-free standard medium. Protein-free standard medium conditioned by human keratinocytes is collected every 24-48 hours for 4-5 days until the cells reach 90-95% confluency. Keratinocyte conditioned medium (CM) is collected and frozen at -80°C. CM can be later thawed, pooled and concentrated. In addition to the original components of protein-free standard medium, concentrated keratinocyte- derived CM contains kdCMF.
  • CM Keratinocyte conditioned medium
  • kdCMF The bioactivity of kdCMF is assayed on any cell type which responds to the factors present in kdCMF.
  • AKR-2B NR6, human dermal fibroblasts, Balb/MK, and human keratinocytes at clonal density are used to detect kdCMF bioactivity (Cook et al., J Cell Phvsiol (1991) 146:277- 289) .
  • the instant invention is shown and described herein in what is considered to be the most practical and preferred embodiments with respect to the growth factor mixtures, their methods of preparation, formulation and use. It is recognized, however, that departures may be made therefrom which are within the scope of the invention and that modifications will occur to one skilled in the art upon reading this disclosure.

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

Abstract

Dans cette invention, on cultive des cellules épithéliales d'origine humaine dans un milieu standard ne contenant pas de protéines pour produire des facteurs de croissance à autocrine et paracrine. Le mélange résultant obtenu à partir de facteurs de milieux conditionnés dérivés des kératinocytes (FMCdk) est utilise pour activer la guérison des plaies superficielles, des ulcérations et d'autres pathologies cutanées hypoprolifératives.
PCT/US1991/006161 1991-08-26 1991-08-28 Milieu conditionne derive des keratinocytes permettant de produire des facteurs de croissance WO1993004164A1 (fr)

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US74847191A 1991-08-26 1991-08-26
US748,471 1991-08-26

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997021442A1 (fr) * 1995-12-11 1997-06-19 University Of Miami Composition stimulant la croissance des cheveux
WO2000069449A3 (fr) * 1999-05-14 2001-02-08 Advanced Tissue Sciences Inc Compositions de milieu de culture cellulaire conditionne et techniques d'utilisation
US6326194B1 (en) * 1993-03-11 2001-12-04 Geoffrey Michael Curtin Method for providing cell growth
US7052684B2 (en) 1995-08-09 2006-05-30 Renovo Limited Methods of healing wounds and fibrotic disorders using IL-10
WO2006026652A3 (fr) * 2004-08-30 2006-08-03 Iken Tissue Therapeutics Inc Compositions et procedes faisant intervenir des proteines wnt pour favoriser la reparation de tissus endommages
US7160726B2 (en) 2001-06-07 2007-01-09 Skin Medica, Inc. Compositions comprising conditioned cell culture media and uses thereof
WO2007005659A3 (fr) * 2005-07-01 2007-04-26 Elfamed Inc Procedes et compositions pour cultiver des keratinocytes
US7566446B2 (en) 1996-12-04 2009-07-28 Renovo Limited Wound healing and treatment of fibrosis

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4423145A (en) * 1981-05-07 1983-12-27 Stampfer Martha R Enhanced growth medium and method for culturing human mammary epithelial cells

Patent Citations (1)

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US4423145A (en) * 1981-05-07 1983-12-27 Stampfer Martha R Enhanced growth medium and method for culturing human mammary epithelial cells

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
CANCER RESEARCH, Volume 49, issued 01 May 1989, D. BOYD et al., "Examination of the Effects of Epidermal Growth Factor on the Production of Urokinase and the Expression of the Plasminogen Activator Receptor in a Human Colon Cancer Cell Line", pages 2427-2432. *
Growth Factors and Other Aspects of Wound Healing: Biological and Clinical Implications, published 1988, by ALAN R. LISS INC. (NEW YORK), G.G. NEMETH et al., pages 1-17. *
INTERNATIONAL JOURNAL OF TISSUE REACTIVITY, Volume X, Number 6, issued 1988, G.R. GROTENDORST, "Growth Factors as Regulators of Wound Repair", pages 337-344. *
JOURNAL OF CELLULAR PHYSIOLOGY, Volume 138, issued 1989, G.D. SHIPLEY et al., "Growth of Normal Human Keratinocytes and Fibroblasts in Serum-Free Medium is Stimulated by Acidic and Basic Fibroblast Growth Factor", pages 511-518. *
JOURNAL OF CELLULAR PHYSIOLOGY, Volume 140, issued 1989, PITTELKOW et al., "Serum-Free Culture of Normal Human Melanocytes: Growth Kinetics and Growth Factor Requirements", pages 565-576. *
JOURNAL OF SURGICAL RESEARCH, Volume 43, issued 1987, A. BUCKLEY et al., "Epidermal Growth Factor Increases Granulation Tissue Formation Dose Dependently", pages 322-328. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, Volume 85, issued March 1988, M. EISINGER et al., "Growth Regulation of Skin Cells by Epidermal Cell-Derived Factors: Implications for Wound Healing", pages 1937-1941. *
TISSUE CULTURE ASSOCIATION 41ST ANNUAL MEETING, Houston, TX, 10-13 June 1990, Annual Meeting Abstracts, issued 1990, G.D. SHIPLEY, "Culture of Human Cells in Optimized Media", page 24A. *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6326194B1 (en) * 1993-03-11 2001-12-04 Geoffrey Michael Curtin Method for providing cell growth
US7052684B2 (en) 1995-08-09 2006-05-30 Renovo Limited Methods of healing wounds and fibrotic disorders using IL-10
US5888551A (en) * 1995-12-11 1999-03-30 University Of Miami Hair growth stimulating composition
WO1997021442A1 (fr) * 1995-12-11 1997-06-19 University Of Miami Composition stimulant la croissance des cheveux
US7566446B2 (en) 1996-12-04 2009-07-28 Renovo Limited Wound healing and treatment of fibrosis
US8138147B2 (en) 1999-05-14 2012-03-20 Skinmedica, Inc. Conditioned cell culture medium compositions and methods of use
AU772829C (en) * 1999-05-14 2005-04-28 Skinmedica, Inc. Conditioned cell culture medium compositions and methods of use
AU772829B2 (en) * 1999-05-14 2004-05-06 Skinmedica, Inc. Conditioned cell culture medium compositions and methods of use
US6372494B1 (en) * 1999-05-14 2002-04-16 Advanced Tissue Sciences, Inc. Methods of making conditioned cell culture medium compositions
WO2000069449A3 (fr) * 1999-05-14 2001-02-08 Advanced Tissue Sciences Inc Compositions de milieu de culture cellulaire conditionne et techniques d'utilisation
US8361485B2 (en) 1999-05-14 2013-01-29 Skinmedica, Inc. Conditioned cell culture medium compositions and methods of use
US8476231B2 (en) 1999-05-14 2013-07-02 Allergan, Inc. Conditioned cell culture medium compositions and methods of use
US9458486B2 (en) 1999-05-14 2016-10-04 Allergen, Inc. Conditioned cell culture medium compositions and methods of use
US7160726B2 (en) 2001-06-07 2007-01-09 Skin Medica, Inc. Compositions comprising conditioned cell culture media and uses thereof
WO2006026652A3 (fr) * 2004-08-30 2006-08-03 Iken Tissue Therapeutics Inc Compositions et procedes faisant intervenir des proteines wnt pour favoriser la reparation de tissus endommages
WO2007005659A3 (fr) * 2005-07-01 2007-04-26 Elfamed Inc Procedes et compositions pour cultiver des keratinocytes

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