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WO2003030959A1 - Dispositif de reparation de la cornee - Google Patents

Dispositif de reparation de la cornee Download PDF

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Publication number
WO2003030959A1
WO2003030959A1 PCT/GB2002/004494 GB0204494W WO03030959A1 WO 2003030959 A1 WO2003030959 A1 WO 2003030959A1 GB 0204494 W GB0204494 W GB 0204494W WO 03030959 A1 WO03030959 A1 WO 03030959A1
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WO
WIPO (PCT)
Prior art keywords
comeal
repair device
contact lens
cells
stem cells
Prior art date
Application number
PCT/GB2002/004494
Other languages
English (en)
Inventor
Baljeen Dhillon
Katheravelu Kanna Ramaesh
Original Assignee
Btg International Limited
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
Priority claimed from GB0124062A external-priority patent/GB0124062D0/en
Priority claimed from GB0211636A external-priority patent/GB0211636D0/en
Application filed by Btg International Limited filed Critical Btg International Limited
Publication of WO2003030959A1 publication Critical patent/WO2003030959A1/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
    • C12N5/0618—Cells of the nervous system
    • C12N5/0621—Eye cells, e.g. cornea, iris pigmented 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
    • A61L27/3804—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
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem 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
    • A61L27/3839—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 the site of application in the body
    • 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
    • A61L27/3895—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 using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B1/00—Optical elements characterised by the material of which they are made; Optical coatings for optical elements
    • G02B1/04—Optical elements characterised by the material of which they are made; Optical coatings for optical elements made of organic materials, e.g. plastics
    • G02B1/041—Lenses
    • G02B1/043—Contact lenses
    • 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
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/16—Materials or treatment for tissue regeneration for reconstruction of eye parts, e.g. intraocular lens, cornea
    • 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
    • C12N2533/50—Proteins
    • C12N2533/56—Fibrin; Thrombin

Definitions

  • the present invention relates to a corneal repair device for the treatment of corneal lesions.
  • the main causes of damage to the cornea are chemical injury; trauma, which occurs most frequently in agricultural/developing communities; and bacterial infection.
  • long-term deep infection can be the major cause of chronic damage.
  • Infectious diseases such as Herpes or secondary to HIV can be treated with antibiotics but often remain latent in the ganglion cells and the infection repeatedly reoccurs causing further inflammation and damage.
  • the other important source of damage that has become common is secondary to bacterial infectious associated with long-term contact lenses.
  • Corneal damage may also result from Vitamin A deficiency, although this is relatively rare in more developed countries it is a major cause of blindness in areas of South East Asia and South America.
  • Corneal damage can be treated in a number of ways. Probably the most common treatment is epithelial layer ablation followed by treatment with antibiotics and a contacting bandage contact lens for up to six months. Corneal transplantation is also used; this is a relatively costly and complex procedure and, in common with other transplant procedures, there may be problems relating to the supply of donor corneas and with rejection of the transplanted cornea. In particular, impaired corneal epithelial cell regeneration and mucous deficiency may prevent successful corneal transplant. In such cases, the transplant may initially appear to be successful, with a clear corneal epithelium present. However, the donor corneal epithelium is gradually replaced by the recipient's cells, which resemble conjunctiva and include goblet cells and neovascularisation, resulting in comeal haze and vision failure.
  • Failed comeal repair, transplant procedures or persistent epithelial lesions are all associated with the existence of conjunctival epithelial in-growth onto the comeal surface. This is thought to occur because the stem cells that regenerate and maintain the comeal epithelium have been depleted or, in some cases, are totally absent. These stem cells, known as corneal epithelial stem cells or limbal stem cells, are normally resident at the comeal scleral junction, in the basal layer of an area known as the comeal limbus. These cells are believed to exist in an undifferentiated state and are capable of proliferation and self-maintenance, producing large number of terminally differentiated functional progeny that can regenerate the comeal epithelium after injury and then maintain its integrity.
  • Neurotrophic keratopathy Neurotrophic keratopathy (neuronal or ischemic)
  • Amniotic membrane transplantation has proved efficacious in the treatment of corneal lesions.
  • the technique is ineffective in the treatment of total stem cell deficiency and the complexity of the guidelines and protocols involved in the harvesting and preparation of human amniotic membrane have limited its use; there is also a risk of transmitting infection.
  • a composite graft of autologous limbal epithelial cells grown in vitro on amniotic membrane has been used by Tsai et al, New England Journal of Medicine 13;343(2): 86-93 (2000) to re-epithelialise a damaged cornea.
  • the use of amniotic membrane is a limiting factor due to the difficulties in preparing the membrane for human application and infection risk.
  • the membrane is also difficult to seed with cells.
  • WO-A-98/31316 teaches a similar approach to that of Pellegrini et al.
  • a sheet of epithelial cells from any source is transferred to a backing such as a contact lens and then applied to the eye.
  • the document teaches that the cell sheet may be held in place with a glue such as fibrin glue or synthetic polymers. It is clear that the authors of the document did not appreciate the advantages of using a population containing stem cells rather than fully differentiated epithelial cells. It also appears that they did not appreciate the necessity of using epithelial cells of comeal, rather than any other, origin.
  • a comeal repair device comprising a contact lens having a modified surface adapted for culturing limbal stem cells, wherein the modified surface comprises a cell substrate.
  • the invention provides an easy-to-handle comeal repair device which both supports the growing of limbal stem cells in vitro and protects the cells from damage in vivo.
  • the device may be used to repair comeal defects and/or replenish depleted limbal stem cell populations.
  • a particular advantage of the device of the invention is that it avoids the use of human amniotic membrane, which is, as mentioned above, difficult to handle and seed. There may also be a variety of ethical issues associated with the use of amniotic tissue.
  • the device is a contact lens, it can be placed and remain comfortably positioned in the eye over extended periods of time without the»need for sutures.
  • the device can also be easily removed from the eye and may be replaced with a further device of the invention if necessary.
  • (1990) relates to intracorneal implants manufactured from polymers which are used in the manufacture of soft contact lenses, such as copolymers of 2-hydroxyethyl methacrylate and methacrylic acid.
  • the surface of these polymers is modified, for example by treatment with an ammonia plasma, to support the growth of comeal epithelial cells. This means that, when implanted in the eye, epithelium will grow over the surface of the implant.
  • the implant of Sipheia et al does not have epithelial cells on it at the implantation stage; it simply has a modified surface which enables epithelial cells to adhere to it after implantation.
  • the device of Sipheia et al is not suitable for transferring cultured limbal stem cells into the eye because it is an implant rather than a contact lens. It is therefore not suitable for use in patients with limbal stem cell deficiency.
  • Sepheia et al describe an experiment in which they tested their modified surface by culturing comeal epithelial cells on it. The experiment demonstrated that the modification of the polymer surfaces greatly increased the attachment of epithelial cells. However, the experiment was simply done with discs of the polymers, not with contact lenses and it does not appear that any measures were taken to ensure a population of stem cells on the surface.
  • Latkany et al, J. Biomed. Mater. Res., 36, 29-37 (1997) describe work in a similar field to that of Sepheia et al.
  • a polyvinylalcohol copolymer hydrogel was dried and treated with a plasma chosen from argon, acetone-O 2 mixtures and ammonia.
  • Latkany et al teach an implant rather than a contact lens. This device would therefore not be suitable as a comeal repair device as it is not suitable for transferring cultured limbal stem cells into the eye. It would certainly not be suitable for use in patients with limbal stem cell deficiency.
  • WO-A-0078928 discloses therapeutic vehicles of which the surfaces have been modified by plasma polymerisation so as to contain acid functionality.
  • the polymeric films can be obtained from the plasmas of volatile organic compounds (at reduced pressure of 10 "2 mmbar and ideally ⁇ 100°C).
  • the volatile compound is termed "monomer” although 'it may not polymerise in the conventional sense.
  • plasma polymer deposition there is generally extensive fragmentation of the starting monomer or ionised gas and a wide range of the resultant fragments or functional groups are undesirably incorporated into the deposit.
  • a low plasma input power low plasma power/monomer flow rate ratio
  • a contact lens refers to a device that fits into the eye such that it is in contact with the cornea.
  • This may be a conventional contact lens or an ophthalmically acceptable material configured like a contact lens, to conform to the shape of the eye.
  • the contact lens is suitable for extended wear and is preferably a soft contact lens, more preferably a bandage contact lens.
  • Contact lenses made of degradable materials may be also used providing they remain substantially intact during the cell culture process and provide sufficient protection for the seeded cell population in vivo so that healing may be initiated.
  • the modified surface adapted for culturing limbal stem cells ' is the concave surface of the contact lens. This has the advantage that the cells growing on the modified surface form a layer which, since it conforms to the shape of the contact lens, will fit the recipient's eye. This also represents a further distinction from the implants of Sepheia et al and
  • cell substrate refers to a surface suitable for limbal cell attachment and growth.
  • the modified surface of the device should facilitate cell attachment in vitro, it is preferable that it does not significantly impair cellular detachment and/or migration of limbal stem cells from the device in vivo.
  • the cell substrate may comprise a protein such as fibrin, fibronectin, albumin, collagen, hyaluronic acid, any protein similar to these or a mixture of such proteins.
  • a protein such as fibrin, fibronectin, albumin, collagen, hyaluronic acid, any protein similar to these or a mixture of such proteins.
  • One of the most suitable proteins for use in the cell substrate is fibrin and protein mixtures containing fibrin.
  • Particularly suitable coatings include mixtures of fibrin and fibronectin and fibrin glue.
  • the cell substrate may also be provided by other means, such as chemical, ionic or plasma treatments which increase the hydrophobicity and surface energy of the contact lens to provide a substrate suitable for cell attachment, see Baier RE, 1986, "Modification of surfaces to meet bioadhesion design goals: a review", J. Adhesion, 20, 171-186. Plasma treatments are also described in Sepheia et al and Latkany et al as discussed above. A further means of providing a cell substrate is to use methods similar to those described in WO-A-0078928. It is possible to culture comeal epithelial cells on contact lenses which have been coated with plasma polymer or copolymer.
  • WO-A-0078928 produces a plasma polymer or copolymer in which the acid functionality of the acid containing monomer (for example acrylic acid) is largely preserved intact from the plasma gas to the plasma polymer or copolymer deposit.
  • These deposits do contain other functional groups (for example hydroxyls arising from post plasma oxidation) but are described in WO-A- 0078928 as "high acid retention", reflecting the high degree of acid retention from the plasma gas into the plasma polymer film.
  • the cell substrate may therefore be obtainable by plasma polymerisation and have at least 5% acid.
  • references to a surface having "5% acid” means that 5 out of every 100 carbon atoms in the plasma polymer is in an acid type environment. It is preferred that the substrate has been obtained by modification of the surface of the contact lens by plasma polymerisation with a volatile acid, a volatile alcohol, a volatile amine or a volatile hydrocarbon.
  • the surface of the contact lens may have been modified by plasma polymerisation of a monomer preparation which may consist of one or more ethylenically unsaturated organic compounds or which may be an alkane.
  • Examples of ethylemcally unsaturated organic compounds include alkenes, which may contain up to 20 carbon atoms but preferably contains up to 12 carbon atoms, for example 8 carbon atoms, carboxylic acids, alcohols or amines (especially ⁇ , ⁇ -unsarurated carboxylic acids, alcohols and amines).
  • a particularly suitable monomer is an octadiene, for example 1,7-octadiene.
  • Particularly preferred cell substrates comprise plasma co-polymers, for example copolymers which comprise at least one organic monomer with at least one hydrocarbon.
  • the hydrocarbon is an alkene, as outlined above.
  • the cell substrate obtainable by plasma polymerisation may have at least 5% acid but it is preferred that the surface has from 5-20% acid and even more preferred that it has greater than 20% acid.
  • the acid may be provided by acrylic acid but alternatively propionic acid or maleic anhydride may be used (Jenkins et al, Langmuir, 16, 6381-6384 (2000)).
  • One of the most preferred plasma polymerised cell substrates may be formed by coating with a plasma copolymer of an acidic monomer, for example acrylic acid and a hydrocarbon, for example 1,7-octadiene.
  • the acrylic acid is provided at 50-100% and 1,7-octadiene at 0-50% in the feed.
  • the cell substrate contains a relatively low concentration of calcium ions; this is thought to be important in maintaining the stem cells in an undifferentiated state.
  • the concentration of calcium ions in the substrate is not greater than about 20mM, more preferably not greater than lOmM and most preferably less than about lmM.
  • Conventional fibrin glue compositions contain calcium ions in a concentration of about 40mM and therefore when fibrin glue is used as the cell substrate, the calcium ion content will usually be reduced to the levels set out above.
  • the purpose of the device of the present invention is to transfer limbal stem cells to the eye of a patient and therefore the cell substrate may be seeded with a cell population comprising limbal stem cells.
  • the cell population has been cultured on the device for a minimum of 7 days before transfer to the eye of the patient.
  • the cell population may be a mixed population of cells, comprising corneal limbal stem and epithelial cells derived from a limbal biopsy taken from the healthy eye of the patient, related living donor or cadaver.
  • the cell population has been pre-enriched, prior to seeding on the device, to increase the proportion of cells that are capable of producing comeal epithelial cells.
  • An advantage of this pre- enrichment is that limbal stem cells can be selected from a mixed population of cells, these stem cells can then be isolated and further expanded before seeding on the contact lens. This presents an opportunity for autologous grafting in patients who have very few remaining stem cells.
  • a particular advantage of the invention is that the seeded cell population is cultured in vitro on the device of the invention, which ensures that the seeded cells adhere well to the cell substrate and are attached to device at the time it is placed in the eye.
  • the seeded cell population forms a subconfluent layer on the concave surface of the contact lens.
  • the seeded cell population comprises a proportion of cells that maintain the ability for clonal proliferation.
  • Preferably around 10% or more of the seeded cell population comprises limbal stem cells which retain the ability for clonal proliferation in vitro.
  • a proportion of limbal stem cells remain undifferentiated and retain their proliferative capacity when the comeal repair device is transferred to the eye.
  • a further advantage of the device of the present invention is that a batch of lenses can be cultured for each patient, thus allowing for retreatment if necessary.
  • the device of the invention may facilitate comeal repair. It is thought that the stem cells seeded on the contact lens cause epithelial cells to migrate from the host limbal tissue and create a new epithelial layer on the cornea (by growth factor mediated kinesis). The stem cells may also migrate from the contact lens and form a new layer on the cornea. However, whatever the actual mechanism, the process is effective and has the advantages that corneal transplant or other radical surgical intervention is avoided and, unlike a corneal transplant, the procedure can be used to treat patients with limbal epithelial stem cell deficiency.
  • this modified surface of the device should facilitate cell attachment in vitro, it is preferable that it does not significantly impair cellular detachment and/or migration of limbal stem cells from the device in vivo.
  • the modified surface is adapted for the in vivo release of limbal stem cells.
  • the device of the present invention may be manufactured from a conventional contact lens, for example a bandage contact lens, by modifying a surface of the contact lens. Therefore, in a second aspect of the invention there is provided a process for manufacturing a comeal repair device according to the invention, the process comprising modifying the surface of a contact lens to provide a cell substrate on the modified surface.
  • Suitable cell substrates have been described above and thus, for example, the process may comprise coating with a protein or a mixture of proteins or plasma polymerisation with a monomer as described above.
  • a protein composition for example fibrin and fibronectin or fibrin glue
  • the coating thickness is preferably not greater than lOO ⁇ m.
  • a protein composition such as fibrin or fibrin-containing mixtures can be coated onto the lens by applying a solution of the protein or a precursor of the protein to the surface of the contact lens.
  • solutions of fibrinogen and thrombin may be mixed on the lens surface and allowed to react to produce fibrin. If a mixture of fibrin and fibronectin is required, a fibronectin solution may be added to the fibrinogen and thrombin mixtures.
  • the process may included the following steps: i. providing at least one organic monomer; ii creating a plasma of the organic monomer; and iii coating at least one lens surface with the plasma.
  • Suitable monomers are outlined above.
  • the process may include the steps of: i. mixing a selected ratio of acid containing monomer and a hydrocarbon in a gas feed; ii. creating a plasma of said mixture; and iii. coating a contact lens with said plasma to provide a surface polymer/copolymer retaining high acid retention of at least 5%.
  • the surface of the contact lens modified in the process of the invention is the concave surface.
  • the process of the second aspect of the invention may further comprise seeding the modified surface of the contact lens, which comprises the cell substrate, with a cell population comprising limbal stem cells and culturing the cells on the contact lens.
  • the seeding density of epithelial stem cells onto the contact lens may be about 10 4 /ml.
  • the concave surface of a bandage contact lens is seeded with cells comprising limbal stem cells, wherein the limbal stem cells are capable of proliferating and fomiing colonies.
  • the process of the second aspect of the invention may further include the step of culturing the cell population in the presence of fibroblast cells before seeding the cell substrate.
  • the process of the second aspect of the invention may further comprise the steps of: harvesting epithelial stem cells from a donor cornea; isolating and culturing the epithelial stem cells on a fibroblast layer; and removing the cultured epithelial stem cells from the fibroblast layer before transferring them to a treated bandage contact lens.
  • the donor tissue may obtained from a living relative, a cadaver or may be a limbal biopsy taken from the patient (autologous).
  • the comeal repair device of the invention may be used in a method of treating corneal lesions, the method comprising inserting the comeal repair device into the eye of a patient in need of such treatment and removing the comeal repair device after healing of the cornea has commenced.
  • the method may also include the steps of replacing the comeal repair device with a further comeal repair device of the invention and removing the further device once healing has progressed. This step may be repeated with yet further contact lenses of the invention.
  • the patient may be suffering from comeal lesions arising from a number of causes, for example chemical/thermal injuries of the ocular surface; Stevens- Johnson syndrome; multiple surgeries or cryotherapies to the limbal region; contact lens-induced keratopathy or toxic effects from lens-cleaning solutions, aniridia (hereditary), keratitis associated with multiple endocrine deficiency (hereditary), neurotrophic keratopathy (neuronal or ischemic), chronic limbitis, peripheral corneal ulcerative keratitis or pterygium and pseudopterygium.
  • comeal lesions arising from a number of causes, for example chemical/thermal injuries of the ocular surface; Stevens- Johnson syndrome; multiple surgeries or cryotherapies to the limbal region; contact lens-induced keratopathy or toxic effects from lens-cleaning solutions, aniridia (hereditary), keratitis associated with multiple endocrine deficiency (hereditary), neurotrophic keratopathy (neuronal or ische
  • a comeal repair device of the first aspect for use in the treatment of comeal lesions.
  • the invention also provides a comeal repair device of the first aspect of the invention in the preparation of an agent for the treatment of comeal lesions.
  • the comeal lesions may arise from the conditions set out above.
  • Figures 1A, IB and 1C show phase contrast photomicrographs of limbal epithelial cells growing on a contact lens that has been coated with a fibrin substrate.
  • FIGS. 1A and IB show cells reaching near confluence (greater than 90%); a number of distinct cell colonies can also be seen against the background of cells.
  • Figure 1C again shows cells reaching near confluence (greater than 90%),.
  • Example 1 Culturing comeal epithelial stem cells obtained from donor tissue on a fibrin coated bandage contact lens.
  • the explants were treated with 0.25 % trypsin and EDTA for 2 hours and the keratinocytes (epithelial cells) collected in low calcium Green's media, see Table 1, every 30 minutes.
  • the harvested single epithelial cells and stem cells were plated, at a density of 2.7 X 10 4 /cm 2 . onto a growth arrested 3T3-J2 fibroblast feeder layer, as described by Rheinwald et al, Cell 6(3), 331-43 (1975), and cultured in a low calcium Green's media. The media was changed every third day. This primary culture reached 70-80% confluence on day 14. Upon good colony formation the 3T3 fibroblasts were removed by vigorous pippeting. The remaining few cells were removed with 0.01 % trypsin for 15 seconds. Trypsin (0.1%) and EDTA were then added for 2 minutes to release the epithelial cells. The viable cell number was determined using trypan blue and hemocytometer chamber.
  • the TISSEELTM Fibrinogen Powder which contains human fibrinogen, was dissolved (according to the manufacturers instructions) using 1 ml of TISSEELTM solution (bovine aprotonin 3000KIU/ml) to produce a fibrinogen solution containing 100-130 mg of total protein of which 75-114 mg is fibrinogen.
  • TISSEELTM solution bovine aprotonin 3000KIU/ml
  • fibrinogen/Aprotinin mixture was diluted with 1 ml of 0.9% NaCl to produce a stock solution with a total protein content of 50-65 mg/ml and fibrinogen content of 37-57 mg ml.
  • TISSEELTM Thrombin powder was reconstituted using 1ml of 0.9% NaCl to produce a thrombin solution which contains 500IU/ml.
  • Thrombin stock solution was prepared by mixing 50 ⁇ l of the thrombin solution and 250 ⁇ l of Calcium chloride (40 ⁇ M/ml) in 10 ml of 0.9% NaCl. (50 ⁇ l of Fibrinogen mixed with 50 ⁇ l of thrombin stock solution is sufficient to produce a lOO ⁇ M thick fibrin layer on a 1 cm 2 surface.) c.) Coating of contact lenses with fibrin substrate.
  • High water content silicon lenses (Bausch & Lomb, Purevision) were washed several times in buffered saline and allowed to dry before coating with fibrin.
  • 25 ⁇ l of Fibrinogen stock and 25 ⁇ l of thrombin stock were added to the concave surface of the contact lens and gently agitated to mix and produce an even fibrin coating on the surface.
  • the coating was left for 10-15 minutes to polymerise and then stored overnight at 4°C; this resulted in a transparent fibrin membrane approximately lOO ⁇ M thick ready for seeding with epithelial cells. 5. Culture of limbal epithelial cells on the contact lens.
  • the limbal epithelial cells at a concentration of 2 - 4 X 10 4 cells per cm 2 from step 3 above were passaged onto the modified surface of the contact lens at a density of approximately 10,000 cells/cm2.
  • the fibrin coated contact lenses with epithelial cells were incubated in multi-well dish containing a feeder layer of growth arrested 3T3 fibroblast cells. This arrangement ensured that there was no direct contact between the epithelial cells on the contact lens and the feeder layer on the bottom of the muti-well dish. After 7 days culture the cells on the lens were subconfluent (60%) and the device ready for transfer to the eye.
  • Example 2 Culturing comeal epithelial stem cells obtained from donor tissue on a fibrin coated bandage contact lens.
  • Fibrin glue components (fibrinogen and thrombin) were obtained from Edinburgh Blood transfusion services, c. Coating of contact lenses with fibrin substrate.
  • Example 3 Culturing comeal epithelial stem cells obtained from donor tissue on a fibrin coated bandage contact lens. a) Preparation of Fibrinogen stock solution
  • Example 4 Culturing comeal epithelial stem cells obtained from a limbal biopsy on a fibrin coated bandage contact lens.
  • Example 1 The methodology of Example 1 is followed using 2 mm 2 of limbal tissue obtained from a limbal biopsy.
  • Limbal biopsy is performed as follows: - a. Peribulbar anaesthesia using 2% lignocaine and 0.75% Marcaine b. Clean the conjunctiva and cornea with 10% Providone Iodine for 30 seconds. c. Wash the Providone Iodine thoroughly with balanced salt solution. d. Remove 2mm 2 limbal biopsy and cover the wound with the conjunctiva.
  • Example 5 Dispase method of harvesting cells from donor or limbal biopsy tissue.
  • Dissect 2mm explants either from Eye Bank donor material or limbal biopsy. Treat with 2mg/ml dispase in DMEM (calcium free) and 10FCS (10% fetal calf serum) for 3 hours. Harvest the cells after three hours
  • Example 6 Explant culture of donor or limbal biopsy tissue.
  • Example 7 Preparation of a contact lens with a fibronectin and fibrin cell susbtrate. Fibrin was prepared according to step 4 of Example 1 above. Plasma fibronectin (Sigma) was added to the fibrin to a final concentration of 20 ⁇ g/ml.
  • Example 8 Comparison of colony forming efficiency (proliferative capacity) of cells grown on contact lens with different cell substrates.
  • Contact lens modified with cell substrate comprising a mixture of fibrin and fibronectin, prepared according to Example 7.
  • CFE Colony forming efficiency
  • the unmodified contact lens did not support the attachment of epithelial cells.
  • the proliferation of cells on the contact lenses modified with fibrin was prolific. After 7 days the cells were confluent on the fibrin modified lens; the colony forming efficiency of cells grown on fibrin substrate was 12%. The cells on the fibrin and fibronectin coated lens also grew well and retained a better CFE (18%).
  • Acrylic acid was obtained from Aldrich Chemical Co. (UK). The monomer was aliquoted into 5ml batches and stored in a refrigerator until required for use. For each polymerisation, one 5ml aliquot was used and then discarded. Prior to polymerisation the monomer was degassed using several freeze-pump/thaw cycles. Polymerisation was carried out in a cylindrical reactor vessel (of 8cm diameter and 50cm length), evacuated by a two stage rotary pump. Stainless steel flanges were sealed to the glass vessel using viton "O" rings. The contact lenses were placed on a two tier stainless steel tray in the centre of the glass vessel.
  • the plasma was sustained by a radio-frequency (13.56MHz) signal generator and amplifier inductively coupled to the reactor vessel by means of an external copper coil.
  • the base pressure in the reactor prior to polymerisation was always ⁇ 1 x 10 mbar.
  • Acrylic acid was polymerised at a plasma power of 1 W and a total flow rate of
  • Plasma polymers were deposited onto the contact lenses (which were positioned with the concave side facing upwards), and clean silica glass cover slips for X-ray photoelectron spectroscopy (XPS) analysis.
  • the pressure with the monomer flowing was typically 4.0 x 10 "2 mbar.
  • a further co-polymerisation using acrylic acid and 1,7-octadiene was carried out using the same conditions.
  • X-ray Photoelectron Spectroscopy XP spectra were obtained on a VG CLAM 2 photoelectron spectrometer employing Mg K ⁇ x-rays. Survey scan spectra (0-1 lOOeV) and narrow scan spectra of C and O were acquired for each sample using analyser pass energies of 50 and 20eV respectively. Spectra were acquired using Specra 6.0 software (R Unwin Software, Cheshire, UK). Subsequent processing was carried out using Scienta Esea Analysis for Windows (Scienta Instruments, Uppsala, Sweden).
  • the spectrometer was calibrated using the Au 4f 7/2peak position at 84.0 eV, and the separation between the Cls and Fls peak positions in a sample of PTFE measured at 397.2eV, which compares well with the 397.19eV reported by Beamson and Briggs (Beamson and Briggs, eds. High Resolution XPS of Organic Polyers: The Scienta ESCA300 Handbook, John Wiley and Sons, Chichester, UK, 1992).
  • the expanded cells were seeded at a density of 2-4x 10 4 cells/lens onto the concave surface of the plasma coated contact lenses and cultured as set out in step 5 of Example 1 above.
  • the comeal cell growth capabilities were compared using conditioned culture media and the fibroblast feeder layer.

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Abstract

Dispositif de réparation de la cornée qui comporte une lentille de contact à surface modifiée pourvue d'un substrat pour cellules sur lequel une population contenant des cellules souches limbiques peut être cultivée. Ledit dispositif est utile pour traiter des lésions de la cornée, en particulier celles dans lesquelles le patient présente une déficience des cellules souches limbiques.
PCT/GB2002/004494 2001-10-06 2002-10-04 Dispositif de reparation de la cornee WO2003030959A1 (fr)

Applications Claiming Priority (4)

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GB0124062.1 2001-10-06
GB0124062A GB0124062D0 (en) 2001-10-06 2001-10-06 Corneal Graft
GB0211636.6 2002-05-21
GB0211636A GB0211636D0 (en) 2002-05-21 2002-05-21 Corneal repair

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005118778A3 (fr) * 2004-06-03 2006-03-16 Roberto Revoltella Methode d'isolement et d'expansion ex vivo de cellules souches de la cornee humaine et utilisations desdites cellules souches
CN100336567C (zh) * 2004-10-19 2007-09-12 北京科宇联合干细胞生物技术有限公司 医用角膜贴片
US8067233B2 (en) 2004-02-26 2011-11-29 Reliance Life Science Pvt. Ltd. Pluripotent embryonic-like stem cells derived from corneal limbus, methods of isolation and uses thereof
US8187875B2 (en) 2004-02-26 2012-05-29 Reliance Life Sciences Pvt. Ltd. Dopaminergic neurons derived from corneal limbus, methods of isolation and uses thereof
US8338175B2 (en) 2006-02-24 2012-12-25 Reliance Life Sciences Pvt. Ltd. Conjunctival tissue system
WO2014152321A1 (fr) * 2013-03-15 2014-09-25 The Jackson Laboratory Isolement de cellules souches non embryonnaires et leurs utilisations
JP2017522016A (ja) * 2014-06-27 2017-08-10 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア 培養哺乳動物輪部幹細胞、その産生方法及びその使用
CN111560348A (zh) * 2020-07-16 2020-08-21 北京昱龙盛世生物科技有限公司 一种角膜缘上皮干细胞分离培养方法

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WO1998031316A1 (fr) * 1997-01-17 1998-07-23 Celadon Science, Llc Procedes permettant d'accelerer la cicatrisation de plaies liees a la refection de la surface de la cornee
WO2000078928A2 (fr) * 1999-06-23 2000-12-28 Celltran Limited Surface de decollement

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WO1998031316A1 (fr) * 1997-01-17 1998-07-23 Celadon Science, Llc Procedes permettant d'accelerer la cicatrisation de plaies liees a la refection de la surface de la cornee
WO2000078928A2 (fr) * 1999-06-23 2000-12-28 Celltran Limited Surface de decollement

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LATKANY R ET AL: "Plasma surface modification of artificial corneas for optimal epithelialization", JOURNAL OF BIOMEDICAL MATERIALS RESARCH, vol. 36, no. 1, July 1997 (1997-07-01), usa, pages 29 - 37, XP002224793 *
PELLEGRINI G ET AL: "Location and Clonal Analysis of Stem Cells and their Differentiated Progeny in the Human Ocular Surface", THE JOURNAL OF CELL BIOLOGY, ROCKEFELLER UNIVERSITY PRESS, US, vol. 145, no. 4, 17 May 1999 (1999-05-17), pages 769 - 782, XP002192318, ISSN: 0021-9525 *
PELLEGRINI G ET AL: "Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium", LANCET, XX, XX, vol. 349, no. 9057, 5 April 1997 (1997-04-05), pages 990 - 993, XP004267066, ISSN: 0140-6736 *
PELLEGRINI GRAZIELLA ET AL: "p63 identifies keratinocyte stem cells", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE. WASHINGTON, US, vol. 98, no. 6, 13 March 2001 (2001-03-13), pages 3156 - 3161, XP002192317, ISSN: 0027-8424 *
RAMA P ET AL: "AUTOLOGOUS FIBRIN-CULTURED LIMBAL STEM CELLS PERMANENTLY RESTORE THE CORNEAL SURFACE OF PATIENTS WITH TOTAL LIMBAL STEM CELL DEFICIENCY", TRANSPLANTATION, WILLIAMS AND WILKINS, BALTIMORE, MD, US, vol. 9, no. 72, 15 November 2001 (2001-11-15), pages 1478 - 1485, XP001061855, ISSN: 0041-1337 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8067233B2 (en) 2004-02-26 2011-11-29 Reliance Life Science Pvt. Ltd. Pluripotent embryonic-like stem cells derived from corneal limbus, methods of isolation and uses thereof
US8187875B2 (en) 2004-02-26 2012-05-29 Reliance Life Sciences Pvt. Ltd. Dopaminergic neurons derived from corneal limbus, methods of isolation and uses thereof
WO2005118778A3 (fr) * 2004-06-03 2006-03-16 Roberto Revoltella Methode d'isolement et d'expansion ex vivo de cellules souches de la cornee humaine et utilisations desdites cellules souches
CN100336567C (zh) * 2004-10-19 2007-09-12 北京科宇联合干细胞生物技术有限公司 医用角膜贴片
US8338175B2 (en) 2006-02-24 2012-12-25 Reliance Life Sciences Pvt. Ltd. Conjunctival tissue system
WO2014152321A1 (fr) * 2013-03-15 2014-09-25 The Jackson Laboratory Isolement de cellules souches non embryonnaires et leurs utilisations
JP2017522016A (ja) * 2014-06-27 2017-08-10 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア 培養哺乳動物輪部幹細胞、その産生方法及びその使用
CN111560348A (zh) * 2020-07-16 2020-08-21 北京昱龙盛世生物科技有限公司 一种角膜缘上皮干细胞分离培养方法

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