WO2022139573A1 - A corneal-healing device and method for producing the same - Google Patents
A corneal-healing device and method for producing the same Download PDFInfo
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- WO2022139573A1 WO2022139573A1 PCT/MY2021/050121 MY2021050121W WO2022139573A1 WO 2022139573 A1 WO2022139573 A1 WO 2022139573A1 MY 2021050121 W MY2021050121 W MY 2021050121W WO 2022139573 A1 WO2022139573 A1 WO 2022139573A1
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- Prior art keywords
- substrate
- stem cells
- limbal
- corneal
- tissue
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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
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/52—Hydrogels or hydrocolloids
-
- 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
- A61L27/14—Macromolecular materials
- A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
-
- 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
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
-
- 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
- A61L27/3869—Epithelial tissues other than skin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F9/00—Methods or devices for treatment of the eyes; Devices for putting-in contact lenses; Devices to correct squinting; Apparatus to guide the blind; Protective devices for the eyes, carried on the body or in the hand
- A61F9/0008—Introducing ophthalmic products into the ocular cavity or retaining products therein
- A61F9/0017—Introducing ophthalmic products into the ocular cavity or retaining products therein implantable in, or in contact with, the eye, e.g. ocular inserts
-
- 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
-
- G—PHYSICS
- G02—OPTICS
- G02C—SPECTACLES; SUNGLASSES OR GOGGLES INSOFAR AS THEY HAVE THE SAME FEATURES AS SPECTACLES; CONTACT LENSES
- G02C7/00—Optical parts
- G02C7/02—Lenses; Lens systems ; Methods of designing lenses
- G02C7/04—Contact lenses for the eyes
Definitions
- One object of the present invention is to provide a corneal-healing device for treating corneal disorders such as those caused by chemical or physical injury, aging, Steven- Johnson’s syndrome, but not limited thereto.
- one of the preceding objects is met, in whole or in part, by the present invention, in which one of the embodiments of the present invention is a corneal-healing device capable of regenerating limbal stem cells of mammals upon temporarily implanting on a defected cornea comprising: a siloxane-hydrogel substrate which takes the form of a contact lens configured with confluent limbal stem cells and extracellular matrix components thereon.
- the limbal stem cells are configured on concave side of the substrate.
- Another object of the present invention is to provide a method for producing a corneal- healing device with confluent LSCs being sufficiently adhering thereto before implantation.
- the method comprises an air-lifting technique used during culturing of the LSCs on a substrate.
- one of the embodiments of the present invention is a method for producing an autologous corneal-healing device capable of regenerating limbal stem cells of a mammals upon temporarily implanting on a defected cornea, the method comprising the steps of: preparing a siloxane-hydrogel substrate which takes the form of a contact lens; explanting limbal tissue of the mammals onto the substrate and incubating thereof; and culturing limbal stem cells obtained from the limbal tissue on the substrate in the presence of extracellular matrix components and growth medium until confluence, wherein the culturing step is conducted using air-lifting technique such that the stem cells substantially adhere to the substrate.
- the step of incubating the limbal tissue on the substrate is conducted in the presence of 5 to 10 % gaseous carbon dioxide for 20 to 40 minutes.
- Figure 2 shows slit lamp anterior segment photography on rabbit eyes performed at day 1 and up to Week 8 corresponding to data shown in Table 1.
- A a) Before burr procedure to induce LSCD.
- F, f Immediately post-burr for creation of LSCD, note epithelial defect on entire cornea,
- Week 2 in treatment group shows a much smaller area of epithelial defect as compared with control group (g).
- B Week 2 showed relatively avascular cornea, mild haze of the cornea compared to (G) hazy iris texture and neovascularization of cornea surface,
- c Almost complete reepithelialisation of cornea at Week 4.
- the substrate is configured with confluent LSCs thereon.
- the method to configure the substrate with confluent LSCs will be explained in later part of the description.
- the LSCs configured on the substrate can be derived from tissues isolated from the host. Implanting a device with autologous cells can reduce risk of rejection.
- the tissues used can be limbal epithelium or conjunctival epithelium. More preferably, limbal epithelium tissue is used.
- the substrate can be further loaded with pharmaceutical agent beneficial to wound healing. For instance, metal oxide nanoparticles.
- the LSCs are confluent on the substrate for healing purpose.
- the LSCs are 70 to 90 % confluent on the substrate.
- the substrate can be further modified such that the LSCs will not fall off before implantation.
- the substrate can be further coated with extracellular matrix components to enhance cell attachment
- the extracellular matrix components used comprises fibronection, laminin or a combination thereof.
- the method further comprises a step of washing the substrate with saline solution prior to the step of explanting limbal tissue thereon.
- the substrate is preferably stretched to allow limbal tissues to adhere on the flat surface.
- the method further comprises a step of isolating limbal tissue from the mammal.
- This step can be exemplified by conducting epithelial biopsy on superior limbal epithelium tissue and excising the tissue from ipsilateral eye of a rabbit under sedation. It is preferred that each biopsy is washed multiple times with saline solution, more preferably using Dulbecco’s Phosphate-Buffered Saline (DPBS) or the like. Thereinafter, the limbal tissue is placed on the concave side of the substrate and incubated under 35 to 37 °C in a 5 to 10 % gaseous carbon dioxide environment for 20 to 40 minutes.
- DPBS Dulbecco’s Phosphate-Buffered Saline
- the growth medium used comprises basal serum-free medium supplemented with human corneal growth supplement (HCGS). More particularly, the basal serum-free medium used can be obtained from Epilife. Additional growth factor such as epithelial growth factors (EGF), insulin, transferrin, bovine pituitary extract (BPE) and hydrocortisone can be provided therein. It is preferred that the growth medium is replenished or changed every few days until achieving 90 % confluence.
- EGF epithelial growth factors
- BPE bovine pituitary extract
- hydrocortisone hydrocortisone
- the excised epithelium was placed in the well and incubate under 37 °C in a 5 % gaseous carbon dioxide environment for up to 20 to 40 minutes. Cloned limbal stem cells are formed on the substrate. Thereinafter, Epilife medium having human corneal growth supplement (HCGS) is provided into the well followed by incubation under 37 °C in a 5 % gaseous carbon dioxide environment and culture up to 10 to 14 days. The level of growth medium used in the well was carefully adjusted to expose surface of the cells using air-lifting technique. The growth medium was replenished every 2 days until achieving 90 % cell confluence.
- HCGS human corneal growth supplement
- the inclusion criteria for rabbit’s selection include; New Zealand white rabbits, healthy adult rabbits with no obvious physical deformity, male rabbits and their weight should be more than 1.5kg. Meanwhile, the exclusion criteria include: rabbits with prior trauma to the eye, pre-existing corneal opacity and the rabbit has been used for other laboratory research before.
- the cells on the device Prior to implantation, the cells on the device were transduced by adding 1 ml of Epilife medium supplimented with Human Corneal Growth Suppliment (HCGS), 0.2 pl of Polybrene and 0.28 pl of Cignal Lenti. After 24 hours, 0.4 pl of puromycin was added to the cells. After 3 days the cells were observed under fluorescence microscope and confocal microscope to observe for green fluorescent protein (GFP) expression.
- HCGS Human Corneal Growth Suppliment
- puromycin was added to the cells. After 3 days the cells were observed under fluorescence microscope and confocal microscope to observe for green fluorescent protein (GFP) expression.
- GFP green fluorescent protein
- the device was removed after 2 weeks of implantation.
- the cornea and ocular surface was evaluated for epithelial healing, cornea clarity, thickness of the cornea and superficial corneal neovascularization.
- Limbal stem cell disease grading was done according to Table 1.
- Slit lamp anterior segment photography on rabbit eyes were performed at day 1 and Week 4 post implantation.
- Spectral-domain anterior segment optical coherence tomography (AS-OCT) was used to measure the central corneal epithelial thickness (CCET) throughout the experiment. Rabbits were sedated and the central anterior segment of the cornea will be scanned.
- OCT Optical coherence tomography
- LSCD Limbal stem cell deficiency
- OCT Optical coherence tomography
- CO Corneal opacity or haze
- CV Corneal neovascularization
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Abstract
The present invention relates to tissue engineering, more particularly to a corneal-healing device capable of regenerating limbal stem cells of mammals upon temporarily implanting on a defected cornea comprising: a siloxane-hydrogel substrate which takes the form of a contact lens configured with confluent limbal stem cells and extracellular matrix components thereon. Further, there is provided a method for producing an autologous corneal-healing device capable of regenerating limbal stem cells of a mammals upon temporarily implanting on a defected cornea, the method comprising the steps of: preparing a siloxane-hydrogel substrate which takes the form of a contact lens; explanting limbal tissue of the mammals onto the substrate and incubating thereof; and culturing limbal stem cells obtained from the limbal tissue on the substrate in the presence of extracellular matrix components and growth medium until confluence, wherein the culturing step is conducted using air-lifting technique such that the stem cells substantially adhere to the substrate.
Description
A CORNEAL-HEALING DEVICE AND METHOD FOR PRODUCING THE SAME
FIELD OF TECHNOLOGY
The present invention relates generally to tissue engineering, more particularly to a corneal- healing device and method for producing the same.
BACKGROUND OF THE INVENTION
The limbus zone of cornea forms a border between the corneal and conjunctival epithelium and its limbal stem cells (LSCs) are essential in maintenance and repair of an adult cornea as they support the repair and regeneration of corneal epithelial tissue. The LSCs play a critical role in corneal wound healing process. In case of corneal disorders, depletion or absence of LSCs reults in impairment of the corneal wound healing process. There are a few approaches to remedy such deficiency. One of them is the conventional corneal transplantation which has the risk of host rejection and lack of donor. Alternative approach is by using sheet-like cell assemblies in which desired cells are grown on a particular cell culture surface which allows reversal of cell adhesion. As such, an intact cell sheet can be transplanted to the host tissue without the use of scaffold. Another approach is cell transplantation using cells such as corneal epithelial, where stem cells are dissociated, cultivated on a supportive matrix (biosynthetic scaffold) like an amniotic membrane, fibrin gel or biopolymers, and then injected into the desired location in the cornea. The drawback of this approach is the lack of stem cell enrichment as the stem cells used contain heterogeneous cell populations therefore may lead to graft failure.
This invention provides a corneal-healing device which act as a temporary implant for regenerating LSCs of a damaged cornea and a method of producing the same. More
particularly, the implant is derived from a method which enables autologous LSCs being sufficiently adhering to the implant before implantation. Further, the implant is configured with autologous LSCs which minimizes xenobiotic infections while also reduces waiting time for an eligible tissue donor.
SUMMARY OF THE INVENTION
One object of the present invention is to provide a corneal-healing device for treating corneal disorders such as those caused by chemical or physical injury, aging, Steven- Johnson’s syndrome, but not limited thereto.
Another object of the present invention is to provide a temporary corneal-healing device which is implanted onto a damaged cornea to replenish the cornea with LSCs and supports repair. The device will be removed therefrom after a suitable period of time.
At least one of the preceding objects is met, in whole or in part, by the present invention, in which one of the embodiments of the present invention is a corneal-healing device capable of regenerating limbal stem cells of mammals upon temporarily implanting on a defected cornea comprising: a siloxane-hydrogel substrate which takes the form of a contact lens configured with confluent limbal stem cells and extracellular matrix components thereon.
In the preferred embodiment, the limbal stem cells are configured on concave side of the substrate.
In the preferred embodiment, the limbal stem cells are derived autologously.
Another object of the present invention is to provide a method for producing a corneal- healing device with confluent LSCs being sufficiently adhering thereto before implantation.
More particularly, the method comprises an air-lifting technique used during culturing of the LSCs on a substrate.
At least one of the preceding objects is met, in whole or in part, by the present invention, in which one of the embodiments of the present invention is a method for producing an autologous corneal-healing device capable of regenerating limbal stem cells of a mammals upon temporarily implanting on a defected cornea, the method comprising the steps of: preparing a siloxane-hydrogel substrate which takes the form of a contact lens; explanting limbal tissue of the mammals onto the substrate and incubating thereof; and culturing limbal stem cells obtained from the limbal tissue on the substrate in the presence of extracellular matrix components and growth medium until confluence, wherein the culturing step is conducted using air-lifting technique such that the stem cells substantially adhere to the substrate.
Preferably, the method further comprises a step of washing the substrate with saline solution prior to the step of explanting limbal tissue thereon.
Advantageously, the growth medium used in the method comprises basal serum-free medium supplemented with human corneal growth supplement (HCGS).
Preferably, the growth medium further comprises amino acids, vitamins, inorganic salts and other defined components (in basal medium) supplemented with epithelial growth factors (EGF), insulin, transferrin, bovine pituitary extract (BPE) and hydrocortisone (in HCGS).
Preferably, the extracellular matrix component is fibronection, laminin or a combination thereof.
Advantageously, the step of incubating the limbal tissue on the substrate is conducted in the presence of 5 to 10 % gaseous carbon dioxide for 20 to 40 minutes.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention is illustrated, although not limited, by the following description of embodiments made with reference to the accompanying drawings in which:
Figure 1 shows an exemplary method used to produce the corneal-healing device in the present invention.
Figure 2 shows slit lamp anterior segment photography on rabbit eyes performed at day 1 and up to Week 8 corresponding to data shown in Table 1. (A, a) Before burr procedure to induce LSCD. (F, f) Immediately post-burr for creation of LSCD, note epithelial defect on entire cornea, (b) Week 2 in treatment group shows a much smaller area of epithelial defect as compared with control group (g). (B) Week 2 showed relatively avascular cornea, mild haze of the cornea compared to (G) hazy iris texture and neovascularization of cornea surface, (c) Almost complete reepithelialisation of cornea at Week 4.
DETAILED DESCRIPTION OF THE INVENTION
Exemplary, non-limiting embodiments of the invention will be disclosed. However, it is to be understood that limiting the description to the preferred embodiments of the invention is merely to facilitate discussion of the present invention and it is envisioned that those skilled in the art may devise various modifications without departing from the scope of the appended claim.
The present invention provides a corneal-healing device capable of regenerating limbal stem cells (LSCs) of mammals upon temporarily implanting on a defected cornea.
Examples of mammals include but not limited to human, rabbit, monkeys and rats i.e sprague dawley, guinea pig, lewis rats. The term ‘mammal’ or ‘mammals’ can be used interchangeably hereinafter with the term ‘host’ or ‘patient’, including singular and plural forms thereof, to refer a living subject with defected cornea. The device in the preferred embodiment is designed for use on an adult mammal but not limited thereto. It shall be noted that the device may vary slightly in dimension and shape for use on an adolescent. Nonetheless, features of the device are essentially the same.
The device can be implanted or attached onto damaged cornea of a mammal to replenish LSCs thereon and supports repair. It is preferable that the device is derived from material biocompatible to a mammalian system and being chemically inert to biological cell culture. Further, it is preferred that the device is derived from material providing suitable permeability to topical eyedrops and drugs common in the art. Further, the device has the shape that fits the eyes, more particularly limbal zone, of a mammal. In the preferred embodiment, the device comprises a siloxane-hydrogel substrate which takes the form of a contact lens. More preferably, the LSCs are configured on concave side of the substrate. Accordingly, the concave side of the substrate is in contact to ocular surface of a host upon implantation. Due to the shape of the substrate, the device can be maintained on the ocular surface without suture or glue. Accordingly, removal of the device as simple as removing a contact lens without causing further discomfort on the host.
In accordance to the present invention, the substrate is configured with confluent LSCs thereon. The method to configure the substrate with confluent LSCs will be explained in later part of the description. As mentioned in preceding description, the LSCs configured on the substrate can be derived from tissues isolated from the host. Implanting a device with autologous cells can reduce risk of rejection. Advantageously, the tissues used can be limbal epithelium or conjunctival epithelium. More preferably, limbal epithelium tissue is used. Optionally, the substrate can be further loaded with pharmaceutical agent beneficial to wound healing. For instance, metal oxide nanoparticles.
Accordingly, it is advantageous that the LSCs are confluent on the substrate for healing purpose. Preferably, the LSCs are 70 to 90 % confluent on the substrate. In view of this, the substrate can be further modified such that the LSCs will not fall off before implantation. In the present invention, the substrate can be further coated with extracellular matrix components to enhance cell attachment Preferably, the extracellular matrix components used comprises fibronection, laminin or a combination thereof.
The present invention also provides a method for producing the autologous corneal-healing device. As mentioned in preceding description, the method also aims to produce a substrate confluent with LSCs. According to Figure 1, the method comprising the steps of: coating a siloxane-hydrogel substrate which takes the form of a contact lens with an extracellular matrix component; explanting limbal tissue of the mammals onto the substrate and incubating thereof; and culturing limbal stem cells obtained from the limbal tissue on the substrate in the presence of growth medium until confluence, wherein the culturing step is conducted using air-lifting technique such that the stem cells substantially adhere to the substrate.
Preferably, the method further comprises a step of washing the substrate with saline solution prior to the step of explanting limbal tissue thereon. Further, the substrate is preferably stretched to allow limbal tissues to adhere on the flat surface.
The method further comprises a step of isolating limbal tissue from the mammal. This step can be exemplified by conducting epithelial biopsy on superior limbal epithelium tissue and excising the tissue from ipsilateral eye of a rabbit under sedation. It is preferred that each biopsy is washed multiple times with saline solution, more preferably using Dulbecco’s Phosphate-Buffered Saline (DPBS) or the like. Thereinafter, the limbal tissue is placed on the concave side of the substrate and incubated under 35 to 37 °C in a 5 to 10 % gaseous carbon dioxide environment for 20 to 40 minutes.
Thereinafter, growth medium is provided onto the substrate and further culturing the cells thereon under 35 to 37 °C in a 5 to 10 % gaseous carbon dioxide environment until confluence. The growth medium used comprises basal serum-free medium supplemented with human corneal growth supplement (HCGS). More particularly, the basal serum-free medium used can be obtained from Epilife. Additional growth factor such as epithelial growth factors (EGF), insulin, transferrin, bovine pituitary extract (BPE) and hydrocortisone can be provided therein. It is preferred that the growth medium is replenished or changed every few days until achieving 90 % confluence.
In accordance to the preceding description, the culturing step is conducted using air-lifting technique such that the cells substantially adhere to the substrate. Air lifting technique is a method of cell culture by which basal stem cells are grown with their basal surfaces in contact with media, and the top of the cellular layer is exposed to the air. The cells are then lifted and media is changed until the development of certain cell phenotype. During limbal epithelial cell expansion in vitro, air-lifting can increase cellular stratification, enlarge surface cells, trigger cellular differentiation, and increase the trans epithelial barrier.
EXAMPLE
An example of the invention is provided hereinafter, following with results to determine safety and efficacy thereof.
FABRICATION OF CORNEAL-HEALING DEVICE
1-2 mm2 of superior limbal epithelium was excised from ipsilateral eye of a rabbit under sedation. Each biopsy was washed 3 times with Dulbecco’s Phosphate-BufferedSaline (DPBS). Siloxane-hydrogel contact lens (Alcon) are washed in saline solution, stretched and placed in 48-well for use. The lens are then coated with fibronectin diluted with Epilife basal medium followed by incubating under 37 °C in a 5 % gaseous carbon dioxide for 30 minutes. The coated lens are subjected to washing steps for 3 times. The lens are ready to
be used as a substrate for limbal tissue explant. The excised epithelium was placed in the well and incubate under 37 °C in a 5 % gaseous carbon dioxide environment for up to 20 to 40 minutes. Cloned limbal stem cells are formed on the substrate. Thereinafter, Epilife medium having human corneal growth supplement (HCGS) is provided into the well followed by incubation under 37 °C in a 5 % gaseous carbon dioxide environment and culture up to 10 to 14 days. The level of growth medium used in the well was carefully adjusted to expose surface of the cells using air-lifting technique. The growth medium was replenished every 2 days until achieving 90 % cell confluence.
IN-VITRO CELL CHARACTERISATION
Rabbit monoclonal antibodies, which is cornea epithelium specific marker (CK3) and goblet cell marker (MUC5AC) purchased from Abeam, Cambridge, UK. LSCs were seeded on contact lens and fixed with 4% ice-cold paraformaldehyde at room temperature for 30 minutes. The cells were subjected to three times washing steps using DPBS. The cells were incubated with primary antibodies (CK3 or MUC5 AC) and left overnight at 4°C, prior to next incubation with secondary antibodies at 37°C for 2 hours. Before microscopic observation was performed, cells were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) and mounted with anti-fading medium. The stained cells were evaluated using a confocal microscopy system (Nikon AIR, Nikon, Japan) and image analysis software (NIS- Elements Viewer 3.20, Nikon, Japan).
CORNEAL DEFECT IN RABBIT MODEL
The inclusion criteria for rabbit’s selection include; New Zealand white rabbits, healthy adult rabbits with no obvious physical deformity, male rabbits and their weight should be more than 1.5kg. Meanwhile, the exclusion criteria include: rabbits with prior trauma to the eye, pre-existing corneal opacity and the rabbit has been used for other laboratory research before.
Twelve New Zealand white rabbits were kept in a bio-bubble room in Tissue-Engineering Center, UKMMC. The rabbits were tagged with numbers and their ages were recorded. Cornea defect in rabbit eyes were performed under local anaesthesia and sedation in Ophthalmology Department, UKMMC.
TRANSPLANTATION OF THE CORNEAL-HEALING DEVICE
Prior to implantation, the cells on the device were transduced by adding 1 ml of Epilife medium supplimented with Human Corneal Growth Suppliment (HCGS), 0.2 pl of Polybrene and 0.28 pl of Cignal Lenti. After 24 hours, 0.4 pl of puromycin was added to the cells. After 3 days the cells were observed under fluorescence microscope and confocal microscope to observe for green fluorescent protein (GFP) expression.
Once LSCs reach 90% confluency on contact lens surface (around 10-14 days), the device were implanted onto the right side of the rabbit’s eye under topical anaesthesia using sterile forceps. Before implantation, the ocular surface was cleaned with 5% povidone iodine and surgical debridement of irregular cornea and conjunctival neovascularisation were done. Tarsorraphy was performed under sedation to prevent the contact lens from getting dislodged. There were two groups, one group will be treated (T) with device (n=6) and the other group were left untreated (UT) (n=6). Chloramphenicol and Maxidex eye drops will be applied to the operated eyes 3 times per day for 5 weeks. The device were kept in-vivo for up to 12 weeks. The results are showed in Table 1 and 2.
OCULAR SURFACE AND CORNEAL EVALUATION
The device was removed after 2 weeks of implantation. The cornea and ocular surface was evaluated for epithelial healing, cornea clarity, thickness of the cornea and superficial corneal neovascularization. Limbal stem cell disease grading was done according to Table 1. Slit lamp anterior segment photography on rabbit eyes were performed at day 1 and Week 4 post implantation. Spectral-domain anterior segment optical coherence tomography (AS-OCT) was used to measure the central corneal epithelial thickness (CCET)
throughout the experiment. Rabbits were sedated and the central anterior segment of the cornea will be scanned. On Week 12 post implantation, the rabbits were sacrificed followed by histological staining (hematoxylin and eosin) and immunohistochemical analysis on corneal construct using CK3 and MUC5AC antibodies. The results are showed in Figure 2, Table 1 , 2 and 3. Area of epithelial defect was measured by Quick Imaging Measurement Software (QIMS), image-processing software acquired from the Department of Mechanical Engineering, UKM.
Table 1 : Day 1 Ocular Surface and Corneal Evaluation
LSCD: Limbal stem cell deficiency
OCT: Optical coherence tomography
CO: Corneal opacity or haze
CV: Corneal neovascularization
FS: Fluorescein staining
Table 2: Week 4 Ocular Surface and Corneal Evaluation
LSCD: Limbal stem cell deficiency OCT: Optical coherence tomography CO: Corneal opacity or haze CV: Corneal neovascularization
FS: Fluorescein staining
Table 3. Reference note for Table 1 and 2.
The present invention may be embodied in other specific forms without departing from its essential characteristics. The described embodiments are to be considered in all aspects only as illustrative and not restrictive. The scope of the invention is, therefore indicated by the appended claims rather than by the foregoing description. All changes, which come within the meaning and range of equivalency of the claims, are to be embraced within their scope.
Claims
1. A corneal-healing device capable of reintroducing limbal stem cells of mammals upon temporarily implanting on a defected cornea comprising: a siloxane-hydrogel substrate which takes the form of a contact lens configured with confluent limbal stem cells, extracellular matrix component and growth factors thereon.
2. The device according to Claim 1, wherein the limbal stem cells are configured on concave side of the substrate.
3. The device according to Claim 1 or 2, wherein the limbal stem cells are derived from donors preferably autologously.
4. A method for producing a corneal-healing device capable of reintroducing limbal stem cells of a human subject upon temporarily implanting on a defected cornea, the method comprising the steps of: preparing a siloxane-hydrogel substrate which takes the form of a contact lens; explanting limbal tissue of the human subject onto the substrate and incubating thereof; and culturing limbal stem cells obtained from the limbal tissue on the substrate in the presence of extracellular matrix component and growth medium until confluence, wherein the culturing step is conducted using air-lifting technique such that the stem cells substantially adhere to the substrate.
5. The method according to Claim 4, wherein the step of preparing the substrate comprises a step of washing the substrate with saline solution prior to the step of explanting limbal tissue thereon.
6. The method according to Claim 4 or 5, wherein the growth medium used comprises basal serum-free medium supplemented with human corneal growth supplement (HCGS).
7. The method according to Claim 6, wherein the growth medium further comprises amino acids, vitamins, inorganic salts and other defined components (in basal medium) supplemented with epithelial growth factors (EGF), insulin, transferrin, bovine pituitary extract (BPE) and hydrocortisone (in HCGS).
8. The method according to any one of Claims 4 to 7, wherein the extracellular matrix component is fibronection, laminin or a combination thereof.
9. The method according to any one of Claims 4 to 8, wherein the step of incubating the limbal tissue on the substrate is conducted in the presence of 5 to 10 % gaseous carbon dioxide throughout the period of culture.
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| US20050013806A1 (en) * | 2003-04-04 | 2005-01-20 | Chang Min S. | Use of contact lens for corneal cell transplant |
| US20050080484A1 (en) * | 2002-09-13 | 2005-04-14 | Ocular Sciences, Inc. | Devices and methods for improving vision |
| US20070128719A1 (en) * | 2003-05-22 | 2007-06-07 | Tissuetech, Inc. | Isolation and expansion of animal cells in cell cultures |
| US20170233698A1 (en) * | 2014-06-27 | 2017-08-17 | The Regents Of The University Of California | Cultured mammalian limbal stem cells, methods for generating the same, and uses thereof |
| US20180353546A1 (en) * | 2006-08-21 | 2018-12-13 | Regenlab Usa Llc | Cell Preparations For Extemporaneous Use, Useful For Healing And Rejuvenation In Vivo |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050080484A1 (en) * | 2002-09-13 | 2005-04-14 | Ocular Sciences, Inc. | Devices and methods for improving vision |
| US20050013806A1 (en) * | 2003-04-04 | 2005-01-20 | Chang Min S. | Use of contact lens for corneal cell transplant |
| US20070128719A1 (en) * | 2003-05-22 | 2007-06-07 | Tissuetech, Inc. | Isolation and expansion of animal cells in cell cultures |
| US20180353546A1 (en) * | 2006-08-21 | 2018-12-13 | Regenlab Usa Llc | Cell Preparations For Extemporaneous Use, Useful For Healing And Rejuvenation In Vivo |
| US20170233698A1 (en) * | 2014-06-27 | 2017-08-17 | The Regents Of The University Of California | Cultured mammalian limbal stem cells, methods for generating the same, and uses thereof |
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