WO2003030958A1 - Greffe de la cornee - Google Patents

Greffe de la cornee Download PDF

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Publication number
WO2003030958A1
WO2003030958A1 PCT/GB2002/004486 GB0204486W WO03030958A1 WO 2003030958 A1 WO2003030958 A1 WO 2003030958A1 GB 0204486 W GB0204486 W GB 0204486W WO 03030958 A1 WO03030958 A1 WO 03030958A1
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WO
WIPO (PCT)
Prior art keywords
contact lens
cells
acid
lens according
plasma
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Application number
PCT/GB2002/004486
Other languages
English (en)
Inventor
David Haddow
Sheila Macneil
Kevin Shakesheff
Original Assignee
Celltran Limited
The University Of Nottingham
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 GB0219544A external-priority patent/GB2394477B/en
Application filed by Celltran Limited, The University Of Nottingham filed Critical Celltran Limited
Publication of WO2003030958A1 publication Critical patent/WO2003030958A1/fr

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Classifications

    • 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
    • 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
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
    • 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
    • C12N2533/30—Synthetic polymers

Definitions

  • the invention relates to a contact lens which is adapted by the provision of an acid containing surface which is obtainable by plasma polymerisation and the use of said lens in the repair of eye tissue, typically the cornea.
  • the ability to see is dependent on the actions of several structures in and around the eye.
  • light rays are reflected from the object to the cornea.
  • the light rays are bent, refracted and focused by the cornea, lens, and vitreous within the eye ball.
  • the lens functions to ensure that light is focused on the retina.
  • the retina then converts light into electrical impulses which are transmitted through the optic nerve to the brain where the image is perceived.
  • the cornea is a highly organised structure being composed of cells and proteins.
  • the cornea does not contain it own direct blood supply to nourish or protect it from infection. This is because the presence of blood vessels would impair the passage of light to the retina.
  • the cornea is composed of five layers. The first of these is the epithelium.
  • the epithelium is the outer most layer and functions primarily to block the passage for foreign bodies into the eye. It also provides a surface that absorbs oxygen and cell nutrients from tears.
  • Below the epithelium is the so called Bowman's layer. It is composed of collagen and provides an attachment site for the epithelium. Beneath the Bowman's layer is the stroma which comprises cells, collagen and also a high percentage of water. Below the stroma is referred to as Descemet's membrane.
  • This membrane is also composed of collagen but of a different type from that found in the stroma.
  • the collagen is secreted by the endothelial cells which lie below it.
  • the endothelium is the inner most layer of the cornea and functions to keep the cornea clear. It achieves this by removing excess fluid from the stroma to prevent distortion and opaqueness in the cornea.
  • the endothelial cells provide an essential function and if the cells are lost either by disease or trauma they are not replaced. If the endothelial layer is destroyed then the only corrective therapy is corneal transplantation.
  • Fuchs' dystrophy is a progressive disease which is more common in women than in men. Fuchs' dystrophy occurs when endothelial cells are lost resulting in inefficient removal of liquid from the stroma. This causes the cornea to swell and distort vision. Ultimately the epithelial layer begins to swell resulting in abnormal curvature of the eyeball resulting in further distortion of vision. The epithelial swelling also produces scaring on the corneal surface.
  • the initial treatment of the disease is the use of drops, ointments or soft contact lenses to alleviate the symptoms. As the disease progresses the only option is a corneal transplant to restore sight.
  • Iridocorneal Endothelial Syndrome This disease is common in women and results in changes in colour of the iris, swelling of the cornea and the development of glaucoma. The disease is usually only present in one eye.
  • the syndrome is defined by a group of three linked conditions referred to as iris nevus syndrome; Chandler's syndrome or essential iris atrophy.
  • iris nevus syndrome Chandler's syndrome or essential iris atrophy.
  • a common feature of this group of diseases is the migration of endothelial cells off the cornea and onto the iris.
  • the loss of endothelial cells from the cornea results in corneal swelling and distortion of the iris with distortion of vision.
  • the cause of the disease is unknown, the treatment includes the use of medication and ultimately corneal transplantation to control corneal swelling.
  • Keratoconus is a disorder resulting in the progressive thinning of the cornea.
  • the disease begins with a thinning of the middle of the cornea which gradually bulges outward resulting in an abnormal curvature.
  • the disease is thought to have at least four origins. Keratoconus can be inherited since sufferers tend to have a family history of the disease.
  • the disease can also occur as a result of eye injury or as a consequence of other diseases either of the eye (for example, retinitis pigmentosa, vernal kerato conjunctivitis) or other diseases which are not in themselves related to the eye but result in keratoconus, (for example Lebers congenital amaurosis, Ehleres- Danlos syndrome and osteogenesis imperfecta).
  • the cornea will stabilise without causing severe vision problems.
  • the cornea will eventually become too scarred and if this occurs the only therapy is corneal transplantation.
  • a yet further example of a disease resulting in corneal damage is lattice dystrophy which results in the accumulation of amyloid deposits or abnormal protein fibres in the stroma.
  • the result of this is an increase in opaqueness resulting in reduced vision. In severe cases this can result in erosion of the outer epithelial layer resulting in a condition known as epithelial erosion. This results in abnormal cornea curvature resulting in loss of vision. This also results in exposure of nerves causing severe pain.
  • a doctor can prescribe eye drops and ointments to reduce the erosion of the cornea.
  • the scarring becomes severe a corneal transplant may be needed. The replacement of the cornea is often only a temporary measure since the donor cornea has a high risk of contracting the disease within as little as three years. This means that the patient has to undergo a further corneal transplantation with the concomitant discomfort and stress.
  • the cornea can also be damaged through infection by a number of pathogenic agents.
  • ocular herpes is a viral infection caused by herpes simplex virus and is a common cause of corneal blindness.
  • Ocular herpes causes corneal inflammation and can be controlled by using antiviral drugs to inhibit viral replication and thereby reduce the destruction of the epithelial cells which the virus infects.
  • stromal keratitis results which causes the immune system to attack and destroy stromal cells. This condition is much more difficult to treat and ultimately results in scarring of the cornea with loss of vision and ultimately blindness. If the disease progresses to this stage then corneal transplantation is the only therapy.
  • Trachoma is a chronic inflammatory disease caused by a bacterial infection the causative agent of which is Chlamydia trachomati .
  • the disease is a progressive disease which begins in childhood and results in corneal scarring.
  • the scarring results from eyelashes turning in and rubbing against the cornea resulting in corneal damage.
  • the scarring results in severe vision loss and blindness, usually when people are 40 to 50 years old.
  • the disease is more common in the developing world affecting more than a 150 million people and blinding around 6 million per year in Asia and North Africa.
  • the current treatment is the use of antibiotics to treat the infection. However people become re-infected and over the years scar tissue builds up in the cornea resulting in blindness. The only corrective measure then is corneal transplantation.
  • eyes are particularly susceptible to chemical burns caused by acid based chemicals (eg muriatic acid, sulphuric acid found in batteries) and alkali based chemicals (e.g. lime, oven cleaners, ammonia).
  • acid based chemicals eg muriatic acid, sulphuric acid found in batteries
  • alkali based chemicals e.g. lime, oven cleaners, ammonia.
  • the cornea becomes scarred to the extent that the only corrective measure is corneal transplantation.
  • WO00789208 which is incorporated by reference, we disclose therapeutic vehicles which are adapted by the provision of surfaces which contain acid functionality. These vehicles have utility in tissue engineering, particularly in the repair of cutaneous wounds. Advantageously, cells which attach to these surfaces proliferate and detach from the vehicle to invade the surrounding tissue to repair the wound.
  • the surfaces are prepared by a method referred to as plasma polymerisation. Plasma polymerisation is a technique which allows an ultra-thin (eg ca.200nm) cross linked polymeric film to be deposited on substrates of complex geometry and with controllable chemical functionality. As a consequence, the surface chemistry of materials can be modified, without affecting the bulk properties of the substrate so treated.
  • Plasmas or ionised gases are commonly excited by means of an electric field. They are highly reactive chemical environments comprising ions, electrons, neutrals (radicals, metastables, ground and excited state species) and electromagnetic radiation. At reduced pressure, a regime may be achieved where the temperature of the electrons differs substantially from that of the ions and neutrals. Such plasmas are referred to as “cold” or “non-equilibrium” plasmas. In such an environment many volatile organic compounds (eg volatile alcohol containing compounds, volatile acid containing compounds, volatile amine containing compounds, or volatile hydrocarbons, neat or with other gases, eg Ar, have been shown to polymerise (H.K.
  • volatile organic compounds eg volatile alcohol containing compounds, volatile acid containing compounds, volatile amine containing compounds, or volatile hydrocarbons, neat or with other gases, eg Ar, have been shown to polymerise (H.K.
  • plasma polymerisation Yasuda, Plasma Polymerisation, Academic Press, London 1985) coating both surfaces in contact with the plasma and those downstream of the discharge.
  • the organic compound is often referred to as the "monomer”.
  • the deposit is often referred to as "plasma polymer”.
  • the advantages of such a mode of polymerisation potentially include: ultra-thin pin-hole free film deposition; plasma polymers can be deposited onto a wide range of substrates; the process is solvent free and the plasma polymer is free of contamination. Under conditions of low power, plasma polymer films can be prepared which retain a substantial degree of the chemistry of the original monomer.
  • plasma polymerised films of acrylic acid contain the carboxyl group (O'Toole L., Beck A.J., Short R.
  • the low power regime may be achieved either by lowering the continuous wave power, or by pulsing the power on and off (Fraser S., Barton D, Bradley J.W., Short R.D, J. Phys. Chem. B., 2002, 22 (106) 5596-5608).
  • Co-polymerisation of one or more compounds having functional groups with a hydrocarbon allows a degree of control over surface functional group concentrations in the resultant plasma copolymer (PCP) (Beck, A.J, Jones F.R., Short R.D., Polymer 1996, 37(24) 5537-5539).
  • the monomers are ethylenically unsaturated.
  • the functional group compound maybe unsaturated carboxylic acid, alcohol or amine, for example, whilst the hydrocarbon is suitably an alkene.
  • ethylene oxide-type molecules eg. tetraethyleneglycol monoallyl ether
  • perfluoro- compounds i.e. perfluorohexane, hexafluoropropylene oxide
  • hydrophobic/superhydrophobic surfaces This technique is advantageous because the surfaces have unique chemical and physical characteristics.
  • the surface wettability, adhesion and frictional/wear characteristics of the substrate can be modified in a controllable and predictable manner.
  • the percentages refer to the percent of carbon atoms in this type of environment.
  • 20 % acid means that 20 of every one hundred carbons in the plasma polymer is in an acid-type environment.
  • the present application relates to the provision of a structure, typically referred to as a contact lens, which has been treated by plasma polymerisation to provide a surface to which cells can readily attach and detach when the structure is placed against the eye.
  • a contact lens comprising at least one surface wherein said surface is obtainable by plasma polymerisaton.
  • said surface is at least 2% acid.
  • said surface is at least 5% acid.
  • contact lens is not meant to be limiting with respect to the form or composition of the lens, rather it is meant to be descriptive of a structure which is adapted to intimately contact the eye to effect delivery of material attached to the surface (eg cells and/or therapeutic agents) of said lens, to the cornea or associated eye structures.
  • Contact lenses are typically disc shaped with a curvature adapted to fit about the outer surface of the eye ball.
  • said surface has been treated by plasma polymerisation with a volatile acid.
  • said surface has been treated by plasma polymerisation with a volatile amine.
  • said surface has been treated by plasma polymerisation with a mixture of volatile acid and volatile hydrocarbon.
  • said cell culture surface comprises a polymer comprising an acid content of at least 2%.
  • said acid content is 2-20% or 5-20%.
  • said acid content is greater than 20%.
  • the percentages refer to the percent of carbon atoms in this type of environment.
  • the acid content of a contact lens surface is determined by methods herein disclosed and are known in the art. For example, percent acid maybe measured by x-ray photoelectron spectroscopy (XPS).
  • Polymerisable monomers that may be used in the practice of the invention preferably comprise unsaturated organic compounds such as, olefmic carboxylic acids and carboxylates, olefmic amines, olefic alcohols, olefinic nitrile compounds, oxygenated olefins, halogenated olefins and olefmic hydrocarbons.
  • olefins include vinylic and allylic forms.
  • the monomer need not be olefinic, however, to be polymerisable. Cyclic compounds such as cyclohexane, cyclopentane and cyclopropane are commonly polymerisable in gas plasmas by glow discharge methods.
  • Derivatives of these cyclic compounds are also commonly polymerisable in gas plasmas.
  • Particularly preferred are polymerisable monomers containing hydroxyl, a ino or carboxylic acid groups. Of these, particularly advantageous results have been obtained through use of acrylic acid or allyl amine. Mixtures of polymerisable monomers may be used. Additionally, polymerisable monomers may be blended with other gases not generally considered as polymerisable in themselves, examples being argon, nitrogen and hydrogen.
  • the polymerisable monomers are preferably introduced into the vacuum chamber in the form of a vapour. Polymerisable monomers having vapour pressures less than 1.3 x 10 " mbar (1.3 Pa) are not generally suitable for use in the practice of this invention.
  • Polymerisable monomers having vapour pressures of at least 6.6 xl0 2 mbar (6.6 Pa) at ambient room temperature are preferred. Where monomer grafting to plasma pressures of at least 1.3 mbar (130Pa) at ambient conditions are particularly preferred polymerisate deposits is employed, polymerisable monomers having vapour. To maintain desired pressure levels, especially since monomer is being consumed in the plasma polymerisations operation, continuous inflow of monomer vapour to the plasma zone is normally practiced. When non polymerisable gases are blended with the monomer vapour, continuous removal of excess gases is accomplished by simultaneously pumping through the vacuum port to a vacuum source, indeed this is the case when employing polymerisable monomers. Since some non-polymerisable gases are often evolved from glow discharge gas plasmas, it is advantageous to control gas plasma pressure at least in part through simultaneous vacuum pumping during plasma polymerisate deposition on a substrate in the process of this invention.
  • Monomers suited for this invention include, fully saturated and unsaturated carboxylic acid compounds up to 20 carbon atoms. More typically 2-8 carbons.
  • Ethylenically unsaturated compounds (especially ⁇ , ⁇ unsaturated carboxylic acids) including acrylic acid, methacrylic acid. Saturated including ethanoic acid and propanoic acid.
  • compounds that can be plasma polymerised that readily hydrolyse to give carboxylic acid functionalities e.g. organic anhydrides (e.g. maleic anhydride) acyl chlorides may be used.
  • said polymer comprises an acrylic acid monomer with at least 2% or 5% acid content.
  • said acid content is between 2% and 20% or between 5% and 20%.
  • the acid content can be > 20%.
  • said polymer comprises an acid co- polymer.
  • the copolymer is prepared by the plasma polymerisation of an organic carboxylic acid (or anhydride) with a saturated (alkane) or unsaturated (alkene, diene or alkyne) hydrocarbon.
  • the hydrocarbon would be of up to 20 carbons (but more usually of 4- 8).
  • alkanes are butane, pentane and hexane.
  • alkenes are butene and pentene.
  • An example of a diene is 1-7 octadiene-.
  • the co- monomer may also be aromatic-containing e.g. styrene.
  • Co-plasma polymerisation may be carried out using any ratio of acid : hydrocarbon, but will be typically using an acid: hydrocarbon ratio between the limits of 100(acid):0(hydrocarbon) to 20 (acid):80 (hydrocarbon) and any ratio between these limits.
  • Plasma polymerised amines are also within the scope of the invention, for example, fully saturated primary, secondary or tertiary amines (e.g. butyl amine, propyl amine, heptylamine) or unsaturated e,g, allyl amine, which are at least 20 carbons but more typically 4-8 carbons. Amines could be co-polymerised with hydrocarbons as above.
  • the glow discharge through the gas or blend of gases in the vacuum chamber may be initiated by means of an audiofrequency, a microwave frequency or a radiofrequency field transmitted to or through a zone in the vacuum chamber.
  • a radiofrequency (RF) discharge transmitted through a spatial zone in the vacuum chamber by an electrode connected to an RF signal generator.
  • RF signal frequencies starting as low as 50 kHz may be used in causing and maintaining a glow discharge through the monomer vapor.
  • an assigned radiofrequency of 13.56 MHz may be more preferable to use to avoid potential radio interference problems as with examples given later.
  • the power loading should be ⁇ 10 9 J/kg to achieve functional group retention in plasma polymers.
  • the glow discharge need not be continuous, but may be intermittent in nature during plasma polymerisate deposition. Or, a continuous glow discharge may be employed, but exposure of a substrate surface to the gas plasma may be intermittent during the overall polymerisate deposition process. Or, both a continuous glow discharge and a continuous exposure of a substrate surface to the resulting gas plasma for a desired overall deposition time may be employed.
  • the plasma polymerisate that deposits onto the substrate generally will not have the same elemental composition as the incoming polymerisable monomer (or monomers). During the plasma polymerisation, some fragmentation and loss of specific elements or elemental groups naturally occurs. Thus, in the plasma polymerisation of acrylic acid, carboxyl content of the plasma polymerisate is typically lower than would correspond to pure polyacrylic acid.
  • nitrogen content of the plasma polymerisate is typically lower than would correspond to pure polyallylamine.
  • Exposure time to either of these unreacted monomers in the absence of a gas plasma allows for grafting of the monomer to the plasma polymerisate, thereby increasing somewhat the level of the functional group (carboxylic acid or amine) in the final deposit.
  • Time intervals between plasma exposure and grafting exposure can be varied from a fraction of a second to several minutes.
  • said surface is provided by coating at least one surface (e.g. the surface in contact with the eye) with a plasma co-polymer of an acidic monomer.
  • a plasma co-polymer of an acidic monomer for example and not by way of limitation, acrylic acid and a hydrocarbon, for example, 1,7-octadiene.
  • acrylic acid is provided at 50-100% and 1,7-octadiene is provided at 0-50% in the gas feed.
  • the contact lens according to the invention as a cell culture surface.
  • said surface is for use in the culture of mammalian cells, preferably human cells.
  • said surface is for use in the culture of cells derived from the cornea.
  • said cells are selected from the group consisting of: corneal endothelial cells; corneal stromal cells; corneal epithelial cells; corneal stem cells or stem cells with corneal potentiality; embryonic stem cells; or adult stem cells.
  • said cell is autologous.
  • said cell is allogenic.
  • the cells used to seed the contact lens according to the invention may be either derived from self (autologous) or non-self (allogenic).
  • a method to culture mammalian cells on a contact lens comprising the steps of: i) providing a preparation comprising; a) corneal derived cells; b) a contact lens according to the invention and attached thereto, fibroblast feeder cells; c) cell culture medium sufficient to support the growth of said mammalian cells; and ii) providing cell culture conditions which promote the proliferation of said mammalian cells on said contact lens.
  • said medium does not include serum.
  • mammalian cells are human.
  • said fibroblast feeder cells are human.
  • said fibroblast feeder cells are human dermal fibroblasts or human oral fibroblasts.
  • said feeder cells are autologous.
  • the direct culturing of mammalian cells on a contact lens according to the invention under conditions herein disclosed has obvious benefits in corneal repair since the fabrication of said conact lens allows the culturing, implantation and transfer of cells to a corneal lesion to be repaired.
  • the absence of serum and the use of autologous cells also minimizes the transfer of xenobiotic agents (e.g. viral agents, prions) from serum and/or feeder cells used in the culture of mammalian cells.
  • a method to treat an animal, preferably a human, suffering from an eye condition which would benefit from the application of the contact lens according to the invention comprising:
  • said eye condition is selected from the group consisting of the following: Fuchs' dystrophy; keratoconus; lattice dystrophy; map- dot-fmger print dystrophy; iridocorneal endothelial syndrome; iris nevus (Cogan- Reese) syndrome; Chandler's syndrome; essential iris atrophy; Stevens- Johnson syndrome.
  • said eye condition is the result of an infection caused by herpes simplex virus.
  • said eye condition is the result of an infection caused by Chlamydia trachomatis.
  • said eye condition is the result of chemical burns.
  • the lens is seeded with a mammalian cell selected from the group consisting of: corneal endothelial cells; corneal stromal cells; corneal epithelial cells; corneal stem cells or stem cells with corneal potentiality; embryonic stem cells; or adult stem cells.
  • a method to coat a contact lens comprising the steps of: i) providing at least one organic monomer (e.g. acrylic acid); ii) creating a plasma of said organic monomer; and iii) coating at least one lens surface with said plasma.
  • organic monomer e.g. acrylic acid
  • said monomer is an acid monomer source comprising 30-99% acid monomer.
  • said acid monomer source consists of a 100% acid monomer source.
  • said method consists of a 100% acrylic acid source.
  • a method to treat a contact lens comprising the steps of: i) providing a selected ratio of a monomer and a hydrocarbon in a gas feed; ii) creating a plasma of said mixture; iii) bringing into contact a contact lens with said plasma mixture to provide a contact lens comprising a co-polymer.
  • said monomer is an acid monomer and said co-polymer is an acid co-polymer.
  • said plasma is created by means of electrical power input (radio frequency 13.56MHz), coupled by means of a copper coil or bands.
  • the reactor volume is in the range 2- 10 L and the reactor is pumped by means of a double stage rotary pump to a base pressure approaching 10 "4 mbar. In the case of replacing the rotary pump with a turbomolecular pump better base pressures 1 ⁇ can be achieved.
  • the monomer pressure is in the range 10 " mbar to 10 " mbar and the monomer flow rate is 1-20 cm 3 / min.
  • the power would be typically 0.5 -50W continuous wave. Those skilled in the art may adjust these parameters to produce like plasmas by pulsing on the micro or milli second time scales.
  • said acid is acrylic acid and said hydrocarbon is a diene and especially a di-unsaturated alkene, for example 1,7-octadiene.
  • said plasma comprises 50-100% unsaturated acid, for example, acrylic acid and 0-50% hexane or diene, (for example, 1,7-octadiene) in the gas feed.
  • said plasma comprises the following ratios of acid (eg acrylic acid) and hexane or diene(egl,7-octadiene); Acid alkene
  • Plasma Polymerisation of an Acid Monomer Acrylic acid was obtained from Aldrich Chemical Co. (UK). The monomer was aliquated 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 canied out in a cylindrical reactor vessel (of 8cm diameter and 50 cm 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 (13.56 MHz) 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 "3 mbar.
  • Acrylic acid was polymerised using continuous wave plasma powers in the range of 1-lOW and a total flow rates in the range 1-20W seem Plasma polymers were deposited onto the contact lenses (which were positioned with the concave side facing upwards), and clean silica glass cover slips or Al foil for 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 range of power, flow rate and pressure conditions.
  • the contact lenses were removed from the glass reactor and cover slips analysed by XPS.
  • XPS X-ray photoelectron spectroscopy
  • plasma polymers containing amine or alcohol functionality were prepared from allyl amine and allyl alcohol monomers, respectively (Aldrich Chemical Co, UK). Plasma polymers were deposited onto the concave side of contact lenses and clean silica glass coverslips or Al foil for XPS.
  • Copolymerisations using allyl amine or allyl alcohol and 1,7-octadiene were carried out in the same manner.
  • XP spectra were obtained on a NG CLAM 2 photoelectron spectrometer employing Mg K ⁇ x-rays.
  • Survey scan spectra (0-1100 eN) and narrow scan spectra of C and O were acquired for each sample using analyser pass energies of 50 and 20 eN respectively.
  • Spectra were acquired using Spectra 6.0 software (R. Unwin Software, Cheshire, UK). Subsequent processing was canied out using Scienta Esca Analysis for Windows (Scienta Instruments, Uppsala, Sweden).
  • the spectrometer was calibrated using the Au 4f 7/2 peak position at 84.0 eN, and the separation between the C Is and F Is peak positions in a sample of PTFE measured at 397.2 eN, which compares well with the value of 397.19 eN reported by Beamson and Briggs (refG.Beamson and D.Briggs, eds., High Resolution XPS of Organic Polymers: The Scienta ESCA300 Handbook, John Wiley and Sons, Chichester, UK, 1992.). Cell Culture
  • Donor corneas were obtained from Bristol Eye Bank, UK.
  • a cornea donor punch (Banon Donor Punch, Katena Eye Instruments, USA) was used to remove a central 8mm section of the donor cornea. From the remaining peripheral tissue 2mm pieces of tissue were cut from the limbal region. Cells were extracted from this tissue enzymatically using Trypsin and primary cultures were established on a feeder layer of inadiated 3T3 mouse fibroblasts. Cells were cultured in low calcium media - approximately 1/10 th physiological (0.1 -0.2mm) using media such as Green's low calcium media for 10-16 days. At this point cells were used experimentally or for clinical use by seeding onto a plasma coated contact lens.
  • corneal epithelial cells may be obtained using a small 2mm biopsy from an unaffected area of the eye to be treated or from the contralateral eye. (This technique could also be used for allogenic cells but would require immunosuppression of the patient for periods of several months if not longer) .
  • Plasma coated contact lenses were seeded on the concave surface with cultured corneal epithelial cells at a range of cell densities. Cells were cultured on these lenses for periods of time from 1-7 days to establish viability and proliferation of cells on lenses.
  • the MTT-ESTA assay was used to measure dehydrogenase activity as an indicator of metabolic viability. For proliferation cells were digested from the lens using a buffer containing urea and detergent (SDS) to analyse DNA. Results showed that cells survived well and increased in number on these plasma coated lenses for several days.
  • SDS urea and detergent
  • colony forming potential of the cultured cells was examined by harvesting cells from the lens using trypsin and then seeding cells at low density (e.g. 1000 cells per 10cm diameter dish) onto a feeder layer of inadiated 3T3 mouse fibroblasts. Cells were left for 7 days and then cells forming successful colonies were stained using rhodamine and also H&E.
  • alternative strategies for expanding corneal epithelial cells these were initially expanded on growth anested human dermal fibroblasts (which could be autologous or allogeneic in origin) or oral fibroblasts (as before, autologous or allogeneic).
  • autologous fibroblasts obtained by punch biopsy from the oral mucosa or the skin and then trypsinisation of the biopsy and collagenase extraction of the fibroblasts
  • autologous fibroblasts obtained by punch biopsy from the oral mucosa or the skin and then trypsinisation of the biopsy and collagenase extraction of the fibroblasts
  • Fibroblasts can be growth arrested by inadiation (in established use since the early 1980s for expansion of cutaneous keratinocytes for clinical use) or can be growth anested by culture of both fibroblasts and corneal epithelial cells in low calcium media - this permits proliferation of corneal epithelial cells but not of dermal fibroblasts or oral fibroblasts as calcium concentrations of less than 0.3mM effectively anest fibroblast proliferation. Hence a media containing calcium at around 0.15mM will allow epithelial cell expansion on growth arrested fibroblasts. This obviates the need to use gamma inadiation to growth anest fibroblasts.
  • corneal epithelial cells increased in number to the same degree as that seen when cells were grown in the absence of fibroblasts and the presence of serum. Essentially, growth anested fibroblasts are substituting for 10% fetal calf serum in this situation.
  • corneal cells were grown to relatively high density on the plasma coated contact lens using one of the methodologies as described.
  • An in vitro corneal wound model was used consisting of a de-epithelialised cornea. Corneas obtained from the Bristol Eye Bank (UK) were treated with 1 molar sodium chloride at 4° overnight and then a scalpel was used to lightly cut through the cornea into the stroma describing a hexagon to encompass a central area of approximately 1cm diameter. Fine forceps were then used to gentle peel the corneal epithelium from the underlying stroma. In developing this model the removal of the epithelium was assessed by staining the cornea with MTT-ESTA - successful removal of the cornea could be readily seen using this technique which allows ready visualisation of metabolically active cells.
  • the bandage contact lens (containing epithelial cells on its inner surface) was then fixed in place using either tissue glue or sutures.
  • the cornea with lens in place could be cultured both at an air- liquid interface and in a submerged culture for 5 days.
  • the glue or sutures were removed using scissors and the contact lens gently removed from the cornea.
  • the transfer of corneal epithelial cells from the lens to the cornea was assessed by staining cornea and lens with MTT-ESTA which allowed rapid visualisation of the location of the cells.
  • Histology of the epithelium on the cornea was also undertaken to assess the nature of the transfened epithelium and the extent to which it had formed a secure attachment to the underlying stroma.
  • the transfer of cells was assessed using both a standard serum containing protocol (in which cells were cultured in the presence of 10% fetal calf serum) and also in a serum free protocol in which corneal cells were initially expanded under serum free conditions and then plated onto the plasma coated contact lens in the presence of growth anested fibroblasts (to maintain the mitogenic drive necessary to ensure corneal epithelium proliferation).
  • a standard serum containing protocol in which cells were cultured in the presence of 10% fetal calf serum
  • a serum free protocol in which corneal cells were initially expanded under serum free conditions and then plated onto the plasma coated contact lens in the presence of growth anested fibroblasts (to maintain the mitogenic drive necessary to ensure corneal epithelium proliferation).
  • the source of fibroblasts could be autologous, dermal or oral mucosa fibroblasts.
  • Serum in contrast contains both adhesive (fibronectin and vitronectin) and anti-adhesive proteins (e.g. very large amounts of albumin). Serum also contains a range of platelet-derived mitogens such as platelet- derived growth factor (PDGF), epidermal growth factor (EGF) and transforming growth factor (TGF- ⁇ ) which all stimulate cell proliferation. It is because of these mitogens that serum is extensively used in cell culture. In producing defined media recombinant mitogens can be used.
  • PDGF platelet- derived growth factor
  • EGF epidermal growth factor
  • TGF- ⁇ transforming growth factor

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Abstract

La présente invention concerne une lentille de contact comportant une surface adaptée par polymérisation par plasma, à laquelle des cellules peuvent se fixer, sur lesquelles elles peuvent proliférer et desquelles elles peuvent se détacher, afin de réparer des lésions cornéennes. L'invention concerne également l'utilisation de ladite lentille de contact dans le traitement d'états pathologiques de l'oeil.
PCT/GB2002/004486 2001-10-06 2002-10-03 Greffe de la cornee WO2003030958A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB0124062A GB0124062D0 (en) 2001-10-06 2001-10-06 Corneal Graft
GB0124062.1 2001-10-06
GB0219544.4 2002-08-22
GB0219544A GB2394477B (en) 2002-08-22 2002-08-22 Cell culture

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004040308A1 (fr) * 2002-10-30 2004-05-13 Plasso Technology Ltd Surface de liaison de sucres
WO2005099894A1 (fr) * 2004-04-15 2005-10-27 Plasso Technology Limited Surface a liaison reversible
WO2010084272A1 (fr) * 2009-01-26 2010-07-29 Tecoptique Procédé et dispositif d'impression sur un verre ophtalmique
US8288118B2 (en) 2007-09-19 2012-10-16 Becton, Dickinson And Company Method of analyzing various surface chemistries for culturing a given cell line
CN116492505A (zh) * 2023-05-11 2023-07-28 山东第一医科大学附属眼科医院(山东省眼科医院) 一种人工角膜内皮移植片及其应用

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WO2000078928A2 (fr) * 1999-06-23 2000-12-28 Celltran Limited Surface de decollement

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004040308A1 (fr) * 2002-10-30 2004-05-13 Plasso Technology Ltd Surface de liaison de sucres
WO2005099894A1 (fr) * 2004-04-15 2005-10-27 Plasso Technology Limited Surface a liaison reversible
US8409874B2 (en) 2004-04-15 2013-04-02 Corning Incorported Reversible binding surface
US8288118B2 (en) 2007-09-19 2012-10-16 Becton, Dickinson And Company Method of analyzing various surface chemistries for culturing a given cell line
US8501477B2 (en) 2007-09-19 2013-08-06 Corning Incorporated Kit including substrates with various surface chemistries
US8748120B2 (en) 2007-09-19 2014-06-10 Corning Incorporated Method of selecting a surface chemistry for culturing a given cell line
WO2010084272A1 (fr) * 2009-01-26 2010-07-29 Tecoptique Procédé et dispositif d'impression sur un verre ophtalmique
FR2941398A1 (fr) * 2009-01-26 2010-07-30 Tecoptique Procede et dispositif d'impression par jet d'encre d'un motif sur un verre ophtalmique
CN116492505A (zh) * 2023-05-11 2023-07-28 山东第一医科大学附属眼科医院(山东省眼科医院) 一种人工角膜内皮移植片及其应用
CN116492505B (zh) * 2023-05-11 2023-12-08 山东第一医科大学附属眼科医院(山东省眼科医院) 一种人工角膜内皮移植片及其应用

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