CORNEAL REPAIR DEVICE
1. Field of the invention The present invention relates to a corneal repair device for the treatment of corneal lesions.
2. Background Art
The main causes of damage to the cornea are chemical injury; trauma, which occurs most frequently in agricultural/developing communities; and bacterial infection. In all cases, 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. Finally a newly emerging but increasing type of corneal damage is that secondary to laser refractory treatment. 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.
If the limbal tissue has been severely damaged it may not be able to generate the phenotypically correct comeal epithelial cells, leading to an abnormal epithelial surface and poor healing. Clinically, this pathologic state can be confirmed by detecting conjunctival goblet cells on the comeal surface through the use of impression cytology. There are a number of potential causes of partial or total limbal stem cell deficiency including: Loss of stem cells due to • 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 Hypofunction of stem cells: (one or both eyes can be involved)
• Aniridia (hereditary)
• Keratitis associated with multiple endocrine deficiency (hereditary)
• Neurotrophic keratopathy (neuronal or ischemic)
• Chronic limbitis
• Peripheral comeal ulcerative keratitis
• Pterygium and pseudopterygium
In patients with a severely depleted limbal stem cells re-population of these cells is necessary to properly re-epithelialise the corneal surface and, once healing is complete, to sustain comeal integrity. A number of procedures have been used, with varying degrees of success, to promote comeal healing and/or replenish limbal stem cell populations.
Limbal transplantation using tissue harvested from the healthy eye, or heterologous tissue from a living relative or cadaver donor, can be grafted onto the damaged eye. Whilst this technique, particularly when using autografts, has proved efficacious, the main problem is the amount of limbal tissue that must be removed from a healthy eye. This may leave the healthy eye at higher risk of future ocular surface disease because much of the limbal stem cell complement has been removed. It also means that only a limited number of stem cells are grafted onto the diseased eye and these may not be effective in causing corneal repair.
Amniotic membrane transplantation has proved efficacious in the treatment of corneal lesions. However, 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. Again, 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. A number of workers have used cell culture techniques to produce sheets of comeal epithelial cells for transplant. Pellegrini et al, Lancet 349, 990-993 (1997) cultured limbal biopsy cells in vitro on a fibrin substrate, producing a sheet of differentiated cells suitable for grafting onto a damaged cornea. The technique has been used to treat a limited number of patients with some success. However, the
cell sheet produced using this methodology is very thin and thus difficult to handle and must be detached from the culture vessel by means of enzyme digestion, prior to transfer to a support. The support, such as gauze or a contact lens assembly, is required to store and transport the delicate sheet of cells prior to grafting. The cell sheet is described as being "differentiated" which implies that the number of viable stem cells remaining may be limited. The epithelial cells may also be subject to damage due to enzymatic digestion and handling.
The same group later repeated their work (Rama et al, Transplantation, 72(9), 1478-1485, published 15 November 2001). It is clear from this later publication that they appreciated the importance of maintaining a population of limbal stem cells in the culture. However, the paper does not address the difficulties of handling a sheet of cells.
WO-A-98/31316 teaches a similar approach to that of Pellegrini et al. In this document, 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.
Schwab et al, Tr. Am. Ophth. Soc, XCVE, 891-986 (1999) discloses a number of different approaches to the treatment of comeal injury including the use of an in vitro cultured sheet of corneal epithelial cells. Again, as with the work discussed above, a support such as a contact lens or collagen shield was used to hold the delicate cell sheet following culture. To summarise, prior art comeal repair techniques, particularly those utilising limbal stem cell replenishment techniques, have shown initial promise. However, there are a number of problems with these techniques that have sometimes limited their chance of a success. Additionally, because of the risks and complexity of the prior art procedures, their use has thus far been limited to a
group of patients with very severe comeal disease, who may have already undergone failed surgical intervention, leading to an irreversible loss of visual acuity.
There is a need for an improved comeal repair device that is easy to handle and seed with cells, is capable of maintaining the proliferative capacity of limbal stem cells and can be transferred easily to, and retained by, the damaged cornea. It has now surprisingly been found that conventional contact lenses, although specifically designed to resist cellular attachment and growth, can be modified to provide an excellent surface on which to grow and maintain limbal stem cells. 3. Disclosure of Invention According to a first aspect of the invention there is provided 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.
Thus, 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.
In addition, because 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.
It is known that the surface of polymeric materials can be modified to support cell growth. For example Sipheia et al (Biomat. Art. Cells. Art. Org., 18(5), 643-655
(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.
This differs from the comeal repair device of the present invention in a number of ways. Firstly, unlike the device of the present invention, it is not a contact lens.
Instead it is an implant which remains permanently in the eye.
Secondly, in contrast to the device of the present invention, which is applied to the eye with limbal stem cells already growing on it, 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-O2 mixtures and ammonia. Following this, cells were cultured both on the hydrogels with treated surfaces and on untreated hydrogels as a control. The argon treated surfaces were found to be most effective in supporting cell proliferation. A synthetic cornea was then treated with an argon plasma and implanted. It was demonstrated that limbal epithelial cells could migrate over and adhere to the treated synthetic cornea.
As with Sepheia et al, however, 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. In 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. However, by employing a low plasma input power (low plasma power/monomer flow rate ratio) it is possible to fabricate films with a high degree of functional group retention from the monomer. An example of such a low power/rate ratio is 2W/2.0sccm. However, other relatively low ratios may be used and are known to those skilled in the art. Copolymerisation of acrylic acid with a hydrocarbon, for example 1,7-octadiene, allows a degree of control over surface functional group concentrations in the resultant plasma copolymer. Plasma copolymers can be deposited directly onto most surfaces regardless of geometry.
There is no teaching in WO-A-0078928 that this surface modification technique could be applied to contact lenses and no suggestion that culturing limbal stem cells on the surface of contact lenses would be a desirable objective.
In the context of the present invention 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. Preferably, 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.
It is preferred that 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
Latkany et al, because, if these implants were contoured at all (and their form is not made clear in the prior art) epithelial cells would grow over the convex face of the implant.
In the context of the present invention the term "cell substrate" refers to a surface suitable for limbal cell attachment and growth.
Although 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.
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. The use of a low power/monomer flow ratio as described in 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.
In the context of the present invention, 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.
In particular, 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 α,β-unsarurated carboxylic acids, alcohols and amines). An alkene monomer may be linear or branched, although linear alkenes are preferred, and may contain more than one C=C bond. 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. Ideally, the hydrocarbon is an alkene, as outlined above.
Other compounds may also be used to form plasma polymers, for example ethylamine, heptylamine, methacrylic acid, propanol, maleic anhydride, allyl alcohol and allyl amine.
As mentioned 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. Ideally, the acrylic acid is provided at 50-100% and 1,7-octadiene at 0-50% in the feed. It is preferred that 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. Advantageously, therefore, 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.
It is greatly preferred that 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. Preferably 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. Preferably the seeded cell population forms a subconfluent layer on the concave surface of the contact lens. It is also preferable that 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. Preferably 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 actual mechanism by which the device of the invention may facilitate comeal repair is not certain. 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. Although 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. In a preferred embodiment of the invention 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.
If a protein composition is used, for example fibrin and fibronectin or fibrin glue, the coating thickness is preferably not greater than lOOμm. 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. For example, in the case of fibrin, 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.
When a the surface is modified by plasma polymerisation, 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.
When the lens is coated with a copolymer, 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%.
Usually, 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 104/ml. Usually, 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. Before seeding the device, it is advantageous to expand the cell population and therefore 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.
An advantage of the process of the present invention and of the repair device produced by the process is that the comeal repair device does not come into contact with fibroblast cells, which present a small risk of transmission of infection. 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. Optionally, 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.
In a further aspect of the invention, there is provided 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.
In these aspects of the invention, the comeal lesions may arise from the conditions set out above.
4. Brief description of the figures.
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.
The cells were seeded at a density of 10000 cells/cm2 on the contact lens, which was then immersed in wells plated with growth arrested 3T3 cells. Figures 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.
Magnification is x200.
Figure 1C again shows cells reaching near confluence (greater than 90%),.
Magnification is x300. 5. Detailed description of the embodiments of the invention
Example 1: Culturing comeal epithelial stem cells obtained from donor tissue on a fibrin coated bandage contact lens.
1. Harvesting the tissue containing limbal stem cells. Donor materials
Donor comeal scleral buttons maintained in organ culture, and deemed unsuitable for transplantation due to poor endothelial counts, were obtained from the Manchester Eye bank and used as the source of comeal stem/epithelial cells. These tissues are tested for HIV and hepatitis B by the Eye bank before being released. The central 8 mm of the comeal tissue was removed using a trephine and the resulting comeal-limbal ring dissected mto 2mm explants.
2. Cell isolation from the harvested tissue - Trypsin and EDTA method of single cell isolation.
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.
3. Culture of isolated cells
The harvested single epithelial cells and stem cells were plated, at a density of 2.7 X 104/cm2 . 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.
4. Preparation of the contact lenses - fibrin coating of contact lenses. Fibrin glue sealant TISSEEL™ (Baxter), which is normally used in vascular and neuro surgical procedures, was used to form a fibrin cell substrate, a) Preparation of Fibrinogen stock solution
The TISSEEL™ Fibrinogen Powder, which contains human fibrinogen, was dissolved (according to the manufacturers instructions) using 1 ml of TISSEEL™ solution (bovine aprotonin 3000KIU/ml) to produce a fibrinogen solution containing 100-130 mg of total protein of which 75-114 mg is fibrinogen. To make the fibrinogen stock
solution the 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. b) Preparation of Thrombin stock solution. TISSEEL™ 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 50μl of the thrombin solution and 250 μl of Calcium chloride (40 μM/ml) in 10 ml of 0.9% NaCl. (50μl of Fibrinogen mixed with 50μl of thrombin stock solution is sufficient to produce a lOOμM thick fibrin layer on a 1 cm2 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 μl of Fibrinogen stock and 25 μl 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 lOOμM 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 104cells per cm2 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.
(The device may be maintained in culture for up to another 7 days prior to transfer or maybe cryopreseved if appropriate.)
Example 2: Culturing comeal epithelial stem cells obtained from donor tissue on a fibrin coated bandage contact lens.
Follow the basic procedure set out in Example 1 replacing the fibrin cell substrate, described in step 1 with a fibrin cell substrate prepared as follows: - a) Preparation of Fibrinogen stock solution
Dissolve human fibrinogen (lyophihsed powder form, 225 mg per vial with 50 units of factor XHi) in 5 ml of Tris buffer solution (20 mM). This gives a fibrinogen stock solution with a fibrinogen concentration of 45mg/ml. b) Preparation of Thrombin stock solution.
Thrombin stock solution is prepared by mixing 50μl of thrombin (500 IU/ml solution) and 250 μl of calcium chloride (40 μM /ml = 40mMol) in 10 ml of 0.9% NaCl to produce a stock solution that contains 5 IU/ml thrombin and ImM (1 μM/ml) CaCl2. Note: Fibrin glue components (fibrinogen and thrombin) were obtained from Edinburgh Blood transfusion services, c. Coating of contact lenses with fibrin substrate. Follow the method set out in Example 1.
Example 3: Culturing comeal epithelial stem cells obtained from donor tissue on a fibrin coated bandage contact lens. a) Preparation of Fibrinogen stock solution
Dissolve human fibrinogen (lyophihsed powder form, 225 mg per vial with 50 units of factor XIII) in 5 ml of 0.9 % NaCl. This gives a fibrinogen stock solution with a fibrinogen concentration of 45 mg/ml. b) Preparation of Thrombin stock solution.
Thrombin stock solution is prepared by mixing 50μl of thrombin (500 IU/ml solution) and 250 μl of calcium chloride (40 μmol/ml = 40 mM) in 10 ml of 0.9% NaCl.
Thrombin stock solution will contain 5 IU/ml thrombin and lmM (1 μM/ml) CaCl2. c. Coating of contact lenses with fibrin substrate. Follow the method set out in Example 1.
Example 4: Culturing comeal epithelial stem cells obtained from a limbal biopsy on a fibrin coated bandage contact lens.
The methodology of Example 1 is followed using 2 mm2 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 2mm2 limbal biopsy and cover the wound with the conjunctiva.
Example 5: Dispase method of harvesting cells from donor or limbal biopsy tissue.
The following is a single cell isolation methodology that is used, as an alternative to the method described in step 2 of Example 1.
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.
Place the tissue explant(s), obtained from biopsy or donor tissue, in a 35 mm dish containing proliferation arrested 3T3 fibroblast cells. Add 1/2 ml of culture medium to the dish, incubate at 37° C in 5% CO2. Add another 2 ml of culture media after six hours. Upon good colony formation remove the 3T3 fibroblasts by vigorous pippeting. Harvest the epithelial cells with 0.01% trypsin and EDTA. Passage the epithelial cells onto the concave side of the modified (cell substrate treated) contact lenses at a density of approximately 10,000 cells/cm2.
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 μg/ml.
Example 8: Comparison of colony forming efficiency (proliferative capacity) of cells grown on contact lens with different cell substrates.
The proliferative capacity of cells that had been cultured on contact lenses was compared for the following lenses: -
Untreated high water content silicon contact lens,
Contact lens modified with fibrin cell substrate, prepared according to
Example 1,
Contact lens modified with cell substrate comprising a mixture of fibrin and fibronectin, prepared according to Example 7.
The different contact lenses were seeded with a population limbal epithelial cells and cultured for 7 days. Colony forming efficiency (CFE), which, whilst not a direct measure, provides a useful indicator of the number of stem cells present, was then assessed. Cells were isolated from the contact lens and 1000 of these cells were subcultured in a 60mm dish on a 3T3 fibroblast feeder layer. After 12 days the 3T3 cells were removed and the number of epithelial cell colonies counted.
CFE= Number of colonies present on day 12 x 100% Number of cells plated
As expected, the unmodified contact lens did not support the attachment of epithelial cells. However, 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%).
Table 1: Low Calcium Green's Media.
Example 9: Modification of Contact Lens Surface by Plasma Polymerisation Materials and Methods Plasma Polymerisation
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
12.0sccm. 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"2mbar. A further co-polymerisation using acrylic acid and 1,7-octadiene was carried out using the same conditions.
For all polymerisation and copolymerisations a deposition time of 15 minutes was used. The monomers were allowed to flow for a further 20 minutes after the plasma was extinguished in order to minimise the uptake of atmospheric oxygen by the deposits on exposure to the laboratory atmosphere. The contact lenses were removed from the glass reactor and cover slips analysed by XPS. In order that the reaction vessel and steel sample tray were in a clean condition for each polymerisation, an etch with an oxygen plasma was carried out after each deposition. The oxygen gas was allowed to flow through the reactor at a pressure of lx lO^mbar. The plasma power was 50W and the etch time was one hour.
XPS of a silica glass cover slip that had previously been polymerised with acrylic acid was examined by XPS to confirm that all the acid had been etched away.
X-ray Photoelectron Spectroscopy XP spectra were obtained on a VG CLAM 2 photoelectron spectrometer employing Mg Kα 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).
CellCulture
Cells were harvested from donor tissue and expanded in culture as set out in steps 2 and 3 of Example 1 above.
The expanded cells were seeded at a density of 2-4x 104 cells/lens onto the concave surface of the plasma coated contact lenses and cultured as set out in step 5 of Example 1 above.
Cell proliferation assays were undertaken on the contact lens to indicate the number of cells present on each lens and to give a measure of the proliferation over the culture period. Cells were also taken from the lens and tested for their ability to form colonies, see Example 8 above.
To assess the comeal epithelial cell transfer from a plasma coated contact lens, the corneal cells were isolated and grown on the plasma coated contact lens as above. The plasma coated contact lens was then placed in contact with an artificial comeal wound model consisting of a de-epithelialised cornea. (This was prepared by either mechanical or physical means, which takes off the comeal epithelium but leaves fibroblast in cornea.) After 4-5 days in contact, the contact lens was removed and both the concave plasma coated lens surface and the de-epithelialised cornea surface is assayed using cell proliferation assays to assess the degree of comeal cell transfer from the comeal repair device. The cell density and colony forming assays were performed on coated lenses which will have different plasma polymer compositions (i.e. varying acrylic acid: octadiene ratios in the gas feed).
The comeal cell growth capabilities were compared using conditioned culture media and the fibroblast feeder layer.