WO2011015862A1 - Support cellulaire comprenant des fibroblastes dermiques - Google Patents

Support cellulaire comprenant des fibroblastes dermiques Download PDF

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Publication number
WO2011015862A1
WO2011015862A1 PCT/GB2010/051278 GB2010051278W WO2011015862A1 WO 2011015862 A1 WO2011015862 A1 WO 2011015862A1 GB 2010051278 W GB2010051278 W GB 2010051278W WO 2011015862 A1 WO2011015862 A1 WO 2011015862A1
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Prior art keywords
fibroblast
tissue
isolated
cells
cell
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PCT/GB2010/051278
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English (en)
Inventor
Colin Albert Buchanan Jahoda
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University Of Durham
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Priority to EP10742030A priority Critical patent/EP2461817A1/fr
Priority to JP2012523390A priority patent/JP2013500738A/ja
Publication of WO2011015862A1 publication Critical patent/WO2011015862A1/fr

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Classifications

    • 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
    • A61F2/02—Prostheses implantable into the body
    • A61F2/10—Hair or skin implants
    • A61F2/105—Skin implants, e.g. artificial skin
    • 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
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/40—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing ingredients of undetermined constitution or reaction products thereof, e.g. plant or animal extracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3886—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells comprising two or more cell types
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/60—Materials for use in artificial skin
    • 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
    • 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
    • C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
    • C12N5/0698—Skin equivalents

Definitions

  • the present invention relates to an improved cell support. More particularly the invention relates to a cell support comprising fibroblast cells isolated from hair bearing skin tissue.
  • Tissue engineering has implications with respect to many areas of clinical and cosmetic surgery and relates to the replacement, restoration or repair of damaged or diseased tissues.
  • Tissue engineering has particular application in wound healing in the provision of skin grafts for skin wound repair.
  • Skin is a highly complex organ covering the external surface of the body. Skin is composed of two layers, the dermis and the epidermis.
  • the dermis is primarily formed of connective tissue containing fibroblasts embedded in a matrix of collagen.
  • the epidermis is the outer layer, which is several cells thick.
  • the epidermis is composed primarily of keratinocytes, which make up over 95% of the cell population. The remainder of the cell population is comprised of dendritic cells, such as Langerhans cells and pigmented cells called melanocytes.
  • the epidermis is essentially cellular and non vascular, there being relatively little extra cellular matrix except for the layer of collagen and other proteins beneath the basal layer of keratinocytes.
  • the keratinocytes are involved in providing the epidermal barrier and have reparative and regenerative properties.
  • Skin functions amongst other things, to prevent water loss from the body and to act as a protective barrier against the action of physical, chemical or infectious agents. Loss of skin may result in mortality or morbidity and in the treatment of large wounds, for example burns, it is recommended to restore the barrier function of the skin, for example by resurfacing with autologous skin grafts.
  • tissue engineering and in particular in skin grafting, is the time required for keratinocyte culture formation. Accordingly, there remains a need for improved methods of establishing keratinocyte culture formation.
  • the invention provides a cell support comprising a fibroblast cell feeder layer, characterized in that said fibroblast cells are isolated from hair bearing skin tissue.
  • said support further comprises an epidermal cell culture, such as a keratinocyte cell culture or an epidermal progenitor.
  • an epidermal cell culture such as a keratinocyte cell culture or an epidermal progenitor.
  • fibroblast cells isolated from hair bearing skin tissue are dermal fibroblasts.
  • fibroblast cells isolated from hair bearing skin tissue are characterized as having an increased expression compared to a fibroblast cell isolated from non-hair bearing tissue of at least 5, 10, 15, 20 or 25 markers selected from the group consisting of:
  • fibroblast cells isolated from hair bearing skin tissue are characterized as expressing increased levels, when compared to a fibroblast cell isolated from non-hair bearing tissue, of a protein marker selected from the group consisting of: SPARC and periostin.
  • said hair bearing skin bears actively growing hair follicles.
  • said hear bearing tissue is selected from the group consisting of: scalp tissue, face tissue, pubic tissue.
  • said culture support further comprises a feeder layer support, such as a matrix, dish, a well, a flask or a plate.
  • a feeder layer support such as a matrix, dish, a well, a flask or a plate.
  • said matrix is a collagen matrix.
  • the invention provides a method of epidermal cell culture comprising co- culturing at least one epidermal cell type together with one or more fibroblast cells,
  • said one or more fibroblast cells are isolated from hair bearing skin tissue.
  • said one or more fibroblast cells are provided as a fibroblast feeder layer.
  • fibroblast cells isolated from hair bearing skin tissue are dermal fibroblasts.
  • fibroblast cells isolated from hair bearing skin tissue are characterized as having an increased expression compared to a fibroblast cell isolated from non-hair bearing tissue of at least 5, 10, 15, 20 or 25 markers selected from the group consisting of:
  • said fibroblast cells isolated from hair bearing skin tissue are characterized as expressing increased levels compared to a fibroblast cell isolated from non- hair bearing tissue of a protein marker selected from the group consisting of: SPARC and periostin.
  • said co-culture is in a serum free medium.
  • said epidermal cell is a keratinocyte, an epidermal progenitor cell or a keratinocyte progenitor.
  • said epidermal cells are human epidermal cells.
  • fibroblast cells are human fibroblasts.
  • fibroblast cells isolated from hair bearing skin tissue are dermal fibroblasts.
  • the invention provides a co-culture vessel comprising a fibroblast feeder layer according to the present invention and one or more epidermal cells.
  • said one or more epidermal cells is a keratinocyte, an epidermal progenitor cell, or a keratinocyte progenitor cell.
  • said vessel further comprises a serum free medium.
  • the invention provides a fibroblast cell isolated from hair bearing skin tissue for use as a medicament.
  • the invention provides use of a fibroblast cell isolated from hair bearing skin tissue in the manufacture of a medicament for skin wound healing.
  • said medicament is prepared for administration with one or more epidermal cells.
  • said one or more epidermal cells are keratinocytes.
  • said fibroblast cell is associated with a matrix, such as a collagen matrix.
  • said medicament is prepared for topical administration.
  • the invention provides a method of healing a skin wound comprising applying one or more fibroblast cells isolated from hair bearing skin tissue to the skin wound.
  • said one or more fibroblast cells is applied to a wound bed of the skin wound.
  • the method further comprises applying one or more epidermal cells to the skin wound or to the wound bed of the skin wound.
  • the invention provides a wound healing composition
  • a wound healing composition comprising one or more fibroblast cells isolated from hair bearing skin tissue and a pharmaceutically acceptable carrier.
  • composition further comprising one or more epithelial cells for simultaneous, separate or sequential administration.
  • said one or more epidermal cells are keratinocytes.
  • composition is prepared for topical administration.
  • the invention provides a wound dressing comprising a medicament in accordance with the present invention or a composition in accordance with the present invention.
  • the invention provides use of a culture of fibroblasts isolated from hair bearing skin to obtain a skin derived precursor (SKP), wherein said culture of fibroblasts has been subject to at least one passage.
  • SBP skin derived precursor
  • Figure 1 details a method for the extraction and processing of RNA for microarray analysis.
  • Figure 2 illustrates the results of lmmunohistochemistry and Western analysis of samples from different human dermal fibroblast cultures show that the fibroblasts from hairy skin (DF) express levels of alpha smooth muscle actin less than hair follicle dermal papilla (DP) and dermal sheath (DS) cells, but greater than fibroblasts from non-hairy foreskin, breast and face skin.
  • Figure 3 shows the growth of human epidermal cells (keratinocytes) in association with different supporting human dermal fibroblasts in 2 dimensional culture. The relative growth of the epidermal cells is measured by the strength of the red Rhodamine dye which stains epidermal keratinocytes. Greater epidermal cell growth is seen with supporting hairy dermal fibroblasts compared with dermal fibroblasts from two non-hairy sites, foreskin and breast, and with mouse 3T3 cells.
  • DF hairy skin
  • DP hair follicle dermal papilla
  • DS dermal shea
  • Figure 4 is a graphical representation showing the growth of epidermal cells (keratinocytes) in association with different supporting dermal fibroblasts.
  • the relative growth of the epidermal cells is measured by absorbance of the red Rhodamine dye (by spectrophometer) which stains keratinocytes.
  • Three replicate samples of dermal cells from a region of hairy skin (pubic) support epidermal cells significantly better than dermal cells from three non-hairy skin regions, breast, foreskin and scalp (from a bald individual).
  • Mouse 3T3 cells are also significantly less good at supporting epidermal growth than the hair skin dermal fibroblasts.
  • Figure 5 is a graphical representation showing the growth of epidermal cells (keratinocytes) in association with different supporting dermal cells.
  • the relative growth of the epidermal cells is measured by absorbance of the red Rhodamine dye (by spectrophometer) which stains keratinocytes.
  • Three replicate samples of dermal cells from a region of hairy skin (hairy scalp) support epidermal cells significantly better than dermal cells from three non-hairy skin regions, breast, foreskin and scalp (from a bald individual).
  • Mouse 3T3 cells are also significantly less good at supporting epidermal growth than the hair skin dermal fibroblasts.
  • Figure 6 is a graphical representation showing the growth of epidermal cells (keratinocytes) in association with different supporting dermal fibroblasts.
  • the relative growth of the epidermal cells is measured by absorbance of the red Rhodamine dye (by spectrophometer) which stains keratinocytes.
  • Three replicate samples of dermal cells from a region of hairy skin (beard) support epidermal cells significantly better than dermal cells from three non-hairy skin regions, breast, foreskin and scalp (from a bald individual).
  • Mouse 3T3 cells are also significantly less good at supporting epidermal growth than the hair skin dermal fibroblasts.
  • the inventors have surprisingly identified that a sub-population of dermal fibroblasts, specifically those isolated from hair bearing skin, have improved cell support properties, particularly epidermal support properties, when compared to the cell support properties of fibroblasts from non-hair bearing skin.
  • the present invention provides an improved method of in vitro epidermal culture and propagation.
  • the invention also provides a method of wound healing and re-epithelialisation using the sub-population of dermal fibroblasts.
  • Fibroblasts are involved in the maintenance of the structural integrity of connective tissue. They secrete precursors of the extracellular matrix and collagen.
  • the sub-population of fibroblasts of the present invention are isolated from hair bearing skin tissue.
  • said hair bearing skin is a tissue containing hair follicles, preferably active hair follicles, e.g. follicles active in the hair cycle. More preferably said tissue is isolated from the scalp, the beard area, the neck, the arms or the pubic (axilla) region.
  • Methods of isolating fibroblasts from skin are well known in the art and the sub-population of dermal fibroblasts used in the present invention can be isolated using any of the general methods available.
  • Biological markers allow the identification and characterization of fibroblasts isolated from hair bearing skin tissue.
  • these biological markers can be used to distinguish fibroblasts isolated from hair bearing skin from fibroblasts isolated from non-hair bearing skin, e.g. foreskin, palm skin, plantar skin and breast.
  • Fibroblasts isolated from hair bearing skin show an increase, when compared to a fibroblast isolated from non-hair bearing skin, e.g. foreskin, palm skin, plantar skin, in the level of expression of at least one polypeptide selected from the group consisting of: ZIC1 , LNX1 , EN1 , MAFB, HMCN1 , LGR4, AGTR1 , ITGA1 1 , POSTN, ISLR, DSP, ITGA8, MY01 D, KCNK2, CH25H, KCNJ15, CLIC2, DPT, TMEPAI, FOXD1 , LGR5, SPON1 , HAPLN1 , THBS4,
  • a nucleic acid molecule e.g. an mRNA encoding a polypeptide selected from ZIC1 , LNX1 , EN1 , MAFB, HMCN1 , LGR4, AGTR1 , ITGA1 1 , POSTN, ISLR, DSP, ITGA8, MY01 D, KCNK2, CH25H, KCNJ15, CLIC2, DPT, TMEPAI, F0XD1 , LGR5, SPON1 , HAPLN1 , THBS4, CYP26B1 , WISP1 and IL7.
  • a fibroblast isolated from hair bearing skin shows an increased expression of at
  • fibroblasts isolated from hair bearing skin also show an increase, when compared to a fibroblast isolated from non-hair bearing skin, e.g. foreskin, palm skin, plantar skin, in the level of at least one secreted protein marker selected from the group consisting of: SPARC (Secreted Protein Acidic and Rich in Cysteine) and periostin, or a nucleic acid molecule, e.g. an mRNA encoding SPARC or periostin.
  • SPARC Secreted Protein Acidic and Rich in Cysteine
  • periostin a nucleic acid molecule, e.g. an mRNA encoding SPARC or periostin.
  • a fibroblast isolated from hair bearing skin shows an increased level of extracellular SPARC and / or periostin protein expression, secretion or activity when compared to a fibroblast isolated from non-hair bearing skin.
  • SPARC also known as osteonectin or BM40
  • BM40 bone
  • SPARC also known as osteonectin or BM40
  • the role of SPARC in tissue repair and remodelling has been expertly reviewed by Phan et al 2006 who describes the main activities of SPARC include: modulating cell-ECM interactions, delaying cell-cycle progression, inhibiting proliferation and angiogenesis, and regulating the expression of a number of growth factor and ECM proteins.
  • a fibroblast isolated from hair bearing skin shows increased expression of a least one polypeptide selected from the group consisting of: ZIC1 , LNX1 , EN1 , MAFB, HMCN1 , LGR4, AGTR1 , ITGA1 1 , POSTN, ISLR, DSP, ITGA8, MYO1 D, KCNK2, CH25H, KCNJ15, CLIC2, DPT, TMEPAI, FOXD1 , LGR5, SPON1 , HAPLN1 , THBS4, CYP26B1 , WISP1 and IL7, or a nucleic acid molecule, e.g.
  • an mRNA encoding said polypeptide and an increased expression of a least one polypeptide selected from the group consisting of SPARC and periostin or a nucleic acid molecule, e.g. an mRNA encoding said polypeptide, when compared to a fibroblast isolated from non-hair bearing skin.
  • a fibroblast isolated from hair bearing skin shows increased expression of each polypeptide of the group consisting of ZIC1 , LNX1 , EN1 , MAFB, HMCN1 , LGR4, AGTR1 , ITGA1 1 , POSTN, ISLR, DSP, ITGA8, MY01 D, KCNK2, CH25H, KCNJ15, CLIC2, DPT, TMEPAI, FOXD1 , LGR5, SPON1 , HAPLN1 , THBS4, CYP26B1 , WISP1 and IL7, or increased expression of a nucleic acid molecule, e.g.
  • an mRNA encoding said polypeptide and an increased expression of each polypeptide selected from the group consisting of SPARC and periostin or a nucleic acid molecule, e.g. an mRNA encoding said polypeptide, when compared to a fibroblast isolated from non-hair bearing skin.
  • fibroblast cells isolated from hair bearing skin tissue are capable of stem cell potential after serial passaging in culture as demonstrated by their capacity to form multipotent skin-derived precursors (SKPs).
  • Fibroblast cells isolated from hair bearing skin tissue are further characterized as having an increased stem cell potential compared to a fibroblast cell isolated from non-hair bearing tissue after serial passaging in culture, preferably said stem cell potential is demonstrated by the capacity to said fibroblasts to form multipotent skin-derived precursors (SKPs).
  • a "passage" refers to a round of subculturing. Accordingly, when cells are subcultured, they are referred to as having been passaged.
  • the fibroblasts are human fibroblasts.
  • Fibroblasts may be isolated from hair bearing skin using methods known in the art. For example, fibroblasts may be isolated from hair bearing skin by explant culture, enzymatic dissociation, cell sorting or a combination thereof.
  • Fibroblasts isolated from hair bearing skin in accordance with the present invention can be used to advantageously promote the growth of cells, preferably epidermal cells, both in vitro, e.g. in tissue engineering and cell culture, and in vivo, in epidermal wound repair and dermal regeneration.
  • the cells supported by the fibroblast sub-population in accordance with the present invention are preferably epidermal cells.
  • Said epidermal cells may be differentiated epidermal cells or epidermal progenitor cells.
  • epidermal progenitor cell is meant a multipotent cell having epidermal potential, e.g. a cell capable of differentiating into an epidermal cell.
  • an epidermal cell in accordance with the invention is selected from a keratinocyte, a melanocyte, a Langerhans cell or a Merkels cell.
  • said epidermal cell is an epidermal progenitor, more preferably a cell having keratinocyte, melanocyte, Langerhans cell or Merkel cell potential.
  • the epidermal cell is a keratinocyte, more preferably an epidermal keratinocyte or a corneal keratinocyte.
  • the epidermal cells are mammalian cells, more preferably human epidermal cells.
  • the cell supported by the fibroblast sub population is an embryonic stem cell, a neural progenitor cell or a blood progenitor cell.
  • culture and “cell culture” are used interchangeably refer to the process whereby cells, taken from a living organism, are grown under controlled conditions, preferably in vitro.
  • fibroblasts isolated from hair bearing skin in accordance with the invention are used as a feeder layer to support a cell or cell culture, preferably an epidermal cell culture, as described above.
  • the fibroblast feeder layer is preferably provided on culture support, such as a matrix, dish, a well, a flask or a plate.
  • the invention provides an improved method of epidermal cell culture, which comprises co-culturing at least one epidermal cell type together with one or more fibroblast cells which is isolated from hair bearing skin tissue.
  • the inventors have demonstrated that fibroblasts isolated from hairy skin provide keratinocytes with factors for growth and
  • the cell culture of the invention may be conducted in the presence of an appropriate cell culture medium.
  • Appropriate epidermal and keratinocyte culture medium are known in the art and include for fibroblasts Minimal Essential Medium (Eagles or Dulbecco"s); for keratinocytes, Dulbecco's MEM, Keratinocyte Basal Medium 2 from PromoCell ' s Cryo-SFM, a serum-free cryo-medium, Keratinocyte-SFM, Keratinocyte nutrient MCDB 153 medium or EpiLife ® Medium from Invitrogen.
  • the culture medium is preferably a serum free culture medium.
  • the culture support and method can be used regenerate tissue, for example in the preparation of skin-grafts. Tissues cultured in accordance with the methods and products described herein can be transplanted into patients to initiate wound healing and repair.
  • the invention provides a fibroblast from hair bearing skin tissue for use as a medicament.
  • the invention provides the use of a fibroblast cell isolated from hair bearing skin tissue in the manufacture of a medicament for skin wound healing.
  • the fibroblast cells are associated with a matrix.
  • the matrix is a biocompatible matrix. More preferably, the matrix is formed from polyhydroxy acids, polyorthoesters, polyanhydrides, proteins, polysaccharides, polyphosphazenes or
  • the support is formed from or comprises collagen, e.g. a collagen-glucosaminoglycan support.
  • the invention provides a wound healing composition
  • a wound healing composition comprising one or more fibroblast cells isolated from hair bearing skin tissue.
  • the fibroblast cells are provided with any standard physiologically and pharmaceutically acceptable carrier.
  • the compositions are sterile and contain a therapeutically effective amount of fibroblasts in an amount suitable for administration to a patient. Fibroblasts may be associated with a matrix as described above.
  • the composition further comprises one or more epithelial cells for simultaneous, separate or sequential administration.
  • said one or more epidermal cells are keratinocytes.
  • said epithelial cells are provided in a matrix as described above.
  • pharmaceutically-acceptable carrier means one or more compatible solid or liquid fillers, diluents or encapsulating substances that are suitable for administration into a human.
  • pharmaceutically acceptable preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, cytokines and optionally other therapeutic agents, preferably agents for use in wound healing such as growth factors, peptides, proteolytic inhibitors, extracellular matrix components, steroids and cytokines.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients.
  • physiologically acceptable refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism.
  • a pharmaceutically acceptable carrier includes any conventional carrier, such as those described in Remington's Pharmaceutical Sciences, by E. W. Martin,
  • compositions of the invention can be administered by any conventional route, including injection.
  • the administration may, for example, be topical, intracavity, subcutaneous, or transdermal.
  • Preferably the composition is prepared for topical administration.
  • compositions of the invention are administered in effective amounts.
  • An "effective amount” is the amount of a composition that alone, or together with further doses, produces the desired response.
  • the compositions used in the foregoing methods preferably are sterile and contain an effective amount of the active ingredient for producing the desired response in a unit of weight or volume suitable for administration to a patient.
  • the response can, for example, be measured by measuring the physiological effects of the composition upon the rate of or extent of wound healing.
  • the invention provides a method for the treatment of skin or a skin wound, comprising applying to the skin, skin wound or skin wound bed a fibroblast isolated from hair bearing skin or composition described herein.
  • the method is of use in skin re-epithelialisation.
  • re-epithelialisation relates to the repair, replacement, functional recovery and ultimate regeneration of damaged epithelium inside the body (including skin), or outside the body.
  • Fibroblasts isolated from hair bearing skin according to the invention can thus be used in the manufacture of a medicament for application to a living body, preferably a human.
  • fibroblasts isolated from hair bearing skin according to the invention are used in the manufacture of a medicament for application to a living body, preferably a human.
  • fibroblasts isolated from hair bearing skin according to the invention are used in the manufacture of a medicament for application to a living body, preferably a human.
  • fibroblasts isolated from hair bearing skin according to the invention are used in the
  • wound relates to damaged tissues, preferably damaged skin, where the integrity of the skin or tissue is disrupted as a result from i.e. external force, bad health status, aging, exposure to sunlight, heat or chemical reaction or as a result from damage by internal physiological processes.
  • Wounds where the epidermis is damaged are considered an open wound.
  • Wound healing is the process of regenerating the covering cell layers of a tissue, preferably by re-epithelialisation or reconstruction.
  • fibroblasts isolated from hair bearing skin are administered to wounds with one or more epithelial cells as described herein.
  • the fibroblasts and optionally one or more epithelial cells are administered with one or more other wound healing agents such as growth factors, peptides, proteolytic inhibitors, extracellular matrix components, steroids or cytokines, oxygen donators or vitamins.
  • additional wound healing agent(s) may be administered separately, simultaneously or sequentially. Such combinations may also be used in the manufacture of the medicament.
  • a patient may be administered the fibroblasts isolated from hair bearing skin and the said one or more epithelial cells as a single medicament.
  • the fibroblasts isolated from hair bearing skin and the said one or more epithelial cells may be administered separately.
  • said fibroblasts isolated from hair bearing skin and or said one or more epithelial cells is autologous, i.e. said cells are derived from the individual to be treated or that biological material added to tissue cultures comes from the donor of the cells for tissue culture.
  • the cells may be non-autologous.
  • the invention provides a wound dressing comprising a fibroblast isolated from hair bearing tissue, a composition or a medicament as described herein.
  • wound dressing refers to a dressing for topical application to a wound.
  • the at least fibroblast isolated from hair bearing tissue, a composition or a medicament may be dispersed in or on a solid sheet of wound contacting material such as a woven or nonwoven textile material, or may be dispersed in a layer of foam such as polyurethane foam.
  • RNA from cells was recovered by conventional techniques and hybridized on Affymetrix full human genome arrays.
  • Total RNA was isolated from cells in integra initially using liquid nitrogen to denature the samples.
  • RNA was isolated using the (RNeasy mini kit, Qiagen).
  • RNA samples were prepared for hybridisation using the one cycle cDNA synthesis kit and applied to an Affymetrix human expression array following the manufacturer's instructions. Data were analysed using GeneSpring® * (Silicon Genetics, USA).
  • 443 genes show differences (increase or decrease) at > 2 fold in Hairy Dermal Fibroblasts v Dermal Sheath.
  • 1 130 genes show differences at >2 fold in Hairy Dermal Fibroblasts v Newborn Fibroblasts.
  • hairy fibroblasts Those genes identified as biomarkers of particular interest for fibroblasts isolated from hair bearing skin (“hairy fibroblasts”) are listed in table 1 below.
  • Biomarkers were identified by immunolabelling.
  • Cells were cultured on glass coverslips for 4 days and fixed using 95% (v/v) MeOH : 5% (v/v) acetone for 15 min at -2O 0 C.
  • Slides were then washed with PBS (3 x 5 minutes) and blocked with 3% (wt/v) BSA., then incubated with primary antibody e.g. mouse monoclonal anti-alphaSMA 1:200 (v/v) for 1 hour at room temperature.
  • Slides were then washed with PBS (3 x 5 minutes) and incubated with secondary antibody and DAPIe.g. alexoflour goat anti-mouse 1:500 (v/v) for 1 hour at room temperature.
  • a coculture approach was used to investigate dermal fibroblasts isolated from hair bearing skin (hairy DF cells) for an ability to support keratinocyte proliferation.
  • Cocultures of dermal and epidermal cells were stained with rhodamine B (rhodamine B specifically staining keratinocytes) and eluted stains were quantified at 550 nm.
  • Explants were cultured for 14 days in MEM supplemented with 20% FBS (Sigma), 2 mM L-glutamine (Invitrogen), 100 units/ml penicillin and 100 ⁇ g/ml streptomycin (Sigma) and 250 ⁇ g/ml amphotericin-B (Invitrogen).
  • FBS FBS
  • 2 mM L-glutamine Invitrogen
  • 100 units/ml penicillin and 100 ⁇ g/ml streptomycin Sigma
  • 250 ⁇ g/ml amphotericin-B Invitrogen.
  • Established dermal cell cultures were cultured in identical media as above, but with a reduction in FBS content from 20% to 10% (v/v).
  • Human keratinocytes were cultured in EpilifeTM (Invitrogen) supplemented with Human
  • Keratinocyte Growth Supplement at 1 :100 v/v (Invitrogen) and used at passage 3 for experimentation.
  • each dermal cell type was co-cultured with human keratinocytes (at passage 3).
  • keratinocytes were cultured using growth arrested murine 3T3 cells (3T3 cells were a kind gift from Dr SE James (University of Brighton) in MEM or Green's media.
  • DMEM and Ham's F12 medium in a 3:1 (v/v) ratio supplemented with 10% (v/v) FBS, 10 ng/mL epidermal growth factor (EGF; R&D Systems, City, UK), 0.4 ⁇ g/mL hydrocortisone (Sigma), 10 ⁇ 10 mol/L cholera toxin (Source), 1 .8 ⁇ 10 ⁇ 4 mol/L adenine (Source), 5 ⁇ g/mL insulin (Sigma), 5 ⁇ g/mL transferrin (Source), 2 x 1 0 ⁇ 3 mol/L glutamine (Sigma), 2 * 1 0 ⁇ 7 mol/L triiodothyrionine, 0.625 ⁇ g/mL amphotericin B (Sigma), 100 IU/mL penicillin and 100 ⁇ g/mL streptomycin (Sigma).
  • Rhodamine B was eluted from keratinocytes by incubation with 0.2N NaOH for 30 minutes at room temperature and the extracted dye was analysed at 550 nm using a S2100 Diode array spectrophotometer (Scientific Laboratory Supplies, city, UK).
  • Keratinocyte attachment and proliferation was observed using DF, feeder cell layers, demonstrated by positive rhodamine B staining of keratinocytes (Figure 3). Keratinocytes cultured for eight days in the presence of growth arrested DS and DP cells were observed to form larger colonies than those cultured in the presence of growth arrested fibroblasts or murine 3T3 cells in MEM.
  • Human DF SKPS were generated from DF cells at early passage numbers (2 and 3) and late passage numbers (1 1 and 12) using methods previously described for SKP formation from cells derived from whole fresh dermal tissue (Biernaskie 2006). Briefly, 25,000 cells per ml were seeded in SKP proliferation media (DMEM (Sigma) and F12 (Invitrogen) in a 3:1 ratio and supplemented with 40 ng/ml FGF2 (R&D Systems); 20 ng/ml EGF (Sigma); 2% B27 (Invitrogen)) and cultured for 21 days in a T25 vented flask (Nunc, Scientific Laboratory Supplies). Addition of 1.5% methycellulose (Sigma) to the SKP proliferation media showed no difference in SKP forming capacity.
  • DMEM Sigma
  • F12 Invitrogen

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Abstract

L'invention concerne un support de culture cellulaire comprenant une couche d'alimentation en cellules fibroblastiques, caractérisé en ce que lesdites cellules fibroblastiques sont isolées du tissu formant le cuir chevelu.
PCT/GB2010/051278 2009-08-03 2010-08-03 Support cellulaire comprenant des fibroblastes dermiques WO2011015862A1 (fr)

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EP10742030A EP2461817A1 (fr) 2009-08-03 2010-08-03 Support cellulaire comprenant des fibroblastes dermiques
JP2012523390A JP2013500738A (ja) 2009-08-03 2010-08-03 皮膚線維芽細胞を有する細胞の支持体

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GB0913469.3A GB2483622A (en) 2009-08-03 2009-08-03 Fibroblast feeder cells

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FR2992221A1 (fr) * 2012-06-22 2013-12-27 Centre Nat Rech Scient Modification des effets immunomodulateurs des cellules
WO2015073778A1 (fr) * 2013-11-14 2015-05-21 Dermarche Labs, Llc Mélanges de fibroblastes, et procédés pour leur fabrication et leur utilisation
WO2015095351A1 (fr) 2013-12-19 2015-06-25 Novartis Ag Compositions et formulations d'arnm de la leptine
CN106987557A (zh) * 2017-05-18 2017-07-28 四川大学华西医院 一种人皮肤多能干细胞（SKPs）培养的改良方法
WO2022047486A3 (fr) * 2020-08-27 2022-04-07 Figene, Llc Application dentaire et parodontale de fibroblastes
WO2023060320A1 (fr) * 2021-10-15 2023-04-20 Bod Science Limited Formulation à base de protéine topique

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2992221A1 (fr) * 2012-06-22 2013-12-27 Centre Nat Rech Scient Modification des effets immunomodulateurs des cellules
WO2013190516A1 (fr) * 2012-06-22 2013-12-27 Centre National De La Recherche Scientifique Modification des effets immunomodulateurs des cellules
WO2015073778A1 (fr) * 2013-11-14 2015-05-21 Dermarche Labs, Llc Mélanges de fibroblastes, et procédés pour leur fabrication et leur utilisation
US10940107B2 (en) 2013-11-14 2021-03-09 Dermaforce Holdings, LLC Fibroblast mixtures and methods of making and using the same
WO2015095351A1 (fr) 2013-12-19 2015-06-25 Novartis Ag Compositions et formulations d'arnm de la leptine
CN106987557A (zh) * 2017-05-18 2017-07-28 四川大学华西医院 一种人皮肤多能干细胞（SKPs）培养的改良方法
WO2022047486A3 (fr) * 2020-08-27 2022-04-07 Figene, Llc Application dentaire et parodontale de fibroblastes
WO2023060320A1 (fr) * 2021-10-15 2023-04-20 Bod Science Limited Formulation à base de protéine topique

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