U.S. patent application number 10/317714 was filed with the patent office on 2003-06-05 for pre-fabricated corneal tissue lens and method of corneal overlay to correct vision.
Invention is credited to Perez, Edward.
Application Number | 20030105521 10/317714 |
Document ID | / |
Family ID | 24478269 |
Filed Date | 2003-06-05 |
United States Patent
Application |
20030105521 |
Kind Code |
A1 |
Perez, Edward |
June 5, 2003 |
Pre-fabricated corneal tissue lens and method of corneal overlay to
correct vision
Abstract
This invention relates to a contact lens made of donor corneal
tissue, to a method of preparing that lens, and to a technique of
placing the lens on the eye. The lens is made of donor corneal
tissue that is acellularized by removing native epithelium and
keratocytes. These cells are replaced with human epithelium and
keratocytes to form a lens that has a structural anatomy similar to
human cornea. The ocular lens is used to correct conditions such as
astigmatism, myopia, aphakia, and presbyopia.
Inventors: |
Perez, Edward; (Menlo Park,
CA) |
Correspondence
Address: |
E. Thomas Wheelock
Morrison & Foerster LLP
755 Page Mill Road
Palo Alto
CA
94304-1018
US
|
Family ID: |
24478269 |
Appl. No.: |
10/317714 |
Filed: |
December 11, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10317714 |
Dec 11, 2002 |
|
|
|
09618580 |
Jul 18, 2000 |
|
|
|
6544286 |
|
|
|
|
Current U.S.
Class: |
351/159.07 ;
351/159.46; 351/159.51; 623/906 |
Current CPC
Class: |
A61F 2/145 20130101;
A61L 27/3641 20130101; A61L 27/3683 20130101; A61L 2430/16
20130101; A61L 27/3813 20130101; A61L 27/3839 20130101; A61F 2/142
20130101; A61L 27/3604 20130101 |
Class at
Publication: |
623/5.16 ;
351/161; 623/906 |
International
Class: |
A61F 002/14 |
Claims
I claim as my invention:
1. An ocular lens device comprising: a lens core comprising donor
corneal tissue having a generally convex anterior surface and a
posterior surface, and having replaced keratocytes in said lens
core or replaced epithelial cells covering at least a portion of
said anterior surface.
2. The ocular lens device of claim 1 wherein said posterior surface
is generally concave.
3. The ocular lens device of claim 1 wherein said lens core
comprises acellular corneal tissue.
4. The ocular lens device of claim 1 wherein said lens core
consists essentially of acellular corneal tissue.
5. The ocular lens device of claim 1 wherein said posterior surface
is concave.
6. The ocular lens device of claim 1 wherein said posterior surface
has been subjected to a shaping step.
7. The ocular lens device of claim 6 wherein said posterior surface
is shaped by an ablative laser.
8. The ocular lens device of claim 1 wherein said ocular lens has a
clarity at least 85% of that of human corneal tissue of a
corresponding thickness.
9. The ocular lens device of claim 8 wherein said ocular lens has a
clarity between about 75% and about 100% of that of human corneal
tissue of a corresponding thickness.
10. The ocular lens device of claim 9 wherein said lens core has a
clarity at least 90% of that of human corneal tissue of a
corresponding thickness.
11. The ocular lens device of claim 1 wherein said lens core
comprises donor corneal tissue.
12. The ocular lens device of claim 11 wherein said lens core
comprises human corneal tissue.
13. The ocular lens device of claim 11 wherein said lens core
comprises allogeneic corneal tissue.
14. The ocular lens device of claim 11 wherein said lens core
comprises xenogenic corneal tissue.
15. The ocular lens device of claim 14 wherein said xenogenic lens
core comprises corneal tissue selected from the group consisting of
rabbit, bovine, porcine, and guinea pig corneal tissue.
16. The ocular lens device of claim 11 wherein said lens core
comprises transgenic corneal tissue.
17. The ocular lens device of claim 11 wherein said donor corneal
tissue has a structural surface and said lens core anterior surface
is the structural surface of the donor corneal tissue.
18. The ocular lens device of claim 1 wherein said size and
configuration is selected to be corrective for at least one
selected from the group consisting of astigmatism, myopia, aphakia,
and presbyopia.
19. The ocular lens device of claim 18 wherein said size and
configuration is selected to be corrective for myopia and said
device has a generally circular, flat lens core center region.
20. The ocular lens device of claim 18 wherein said size and
configuration is selected to be corrective for aphakia and said
device has a generally flattened perimeter.
21. The ocular lens device of claim 18 wherein said size and
configuration is selected to be corrective for presbyopia and has
generally circular lens core central region without correction.
22. The ocular lens device of claim 18 wherein said size and
configuration is selected to be bifocal.
23. The ocular lens device of claim 22 wherein said lens core
further comprises an opaque annular ring having a central open
region and peripheral diameter.
24. The ocular lens device of claim 23 wherein said ring is formed
by tatooing or placement of opaque material on said posterior
surface.
25. The ocular lens device of claim 23 wherein said central open
region has a diameter less than about 1.5 mm.
26. The ocular lens device of claim 25 wherein said central open
region has a diameter between about 0.5 mm and about 1.5 mm.
27. The ocular lens device of claim 25 wherein said central open
region has a diameter between about 0.75 mm and about 1.25 mm.
28. The ocular lens device of claim 24 wherein said ring comprises
a Dacron mesh.
29. The ocular lens device of claim 23 wherein said ring peripheral
diameter is between 3-5 mm.
30. The ocular lens device of claim 29 wherein said ring peripheral
diameter is selected to be less than the pupillary diameter of a
selected recipient eye in low light.
31. The ocular lens device of claim 1 wherein said lens core
further contains a therapeutic agent, immunosuppresive agent, or
growth factors.
32. The ocular lens device of claim 1 wherein said lens core has
not been frozen, lyophilized, or chemically treated by a
fixative.
33. The ocular lens device of claim 11 wherein said lens core is
comprised of corneal tissue grown in vitro.
34. The ocular lens device of claim 1 wherein said epithelial cells
and keratocytes comprise human corneal cells.
35. The ocular lens device of claim 34 wherein said epithelial
cells and keratocytes comprise neonatal, fetal, or tissue bank
corneal cells.
36. The ocular lens device of claim 1 wherein said lens core has a
thickness and said thickness is less than 300 .mu.m.
37. The ocular lens device of claim 36 wherein said lens core
thickness is between 5-100 .mu.m.
38. The ocular lens device of claim 1 wherein said lens core has
replaced keratocytes.
39. The ocular lens device of claim 1 wherein said lens core has
replaced epithelial cells covering at least a portion of said
anterior surface.
40. A method for correcting the vision of a human eye having a
cornea with an anterior surface, comprising the steps of: a)
preparing the anterior surface of the cornea; and b) introducing
the ocular device of any of claims 1-39 upon said prepared anterior
surface.
41. The method of claim 40 wherein said preparing step comprises
removing a substantial portion of any epithelial cells present upon
the anterior surface.
42. A method for the preparation of an ocular lens device
comprising: a) harvesting a lens core comprising donor corneal
tissue and having a generally convex anterior surface and a
generally concave posterior surface; and b) having replaced
epithelium covering at least a portion of said generally convex
anterior surface; and c) having replaced keratocytes repopulating
said lens core, comprising the steps of: i) devitalizing said lens
core; and ii) revitalizing said lens core.
43. The method of claim 42 further comprising the step of shaping
said posterior surface.
44. The method of claim 43 wherein said posterior surface is shaped
by an ablative laser.
45. The method claim 43 wherein said posterior surface is shaped by
an excimer laser or other suitable shaping lasers.
46. The method of claim 43 wherein said posterior surface is shaped
by water jet cutting.
47. The method of claim 43 wherein said posterior surface is shaped
to correct at least one selected from the group consisting of
myopia, aphakia, presbyopia, and astigmatism.
48. The method of claim 42 further comprising the step of
harvesting said lens core.
49. The method of claim 48 wherein said lens core is harvested from
human, rabbit, bovine, porcine, or guinea pig corneal tissue.
50. The method of claim 42 wherein said revitalization step
includes cells cultured from human corneal tissue.
51. The method of claim 50 wherein said cells are cultured from
neonatal tissue, fetal tissue, or tissue bank tissue.
52. The method of claim 43 further comprising the step of
sterilizing said lens core after said shaping step.
53. The method of claim 52 wherein said sterilization step includes
the step of contacting said lens core with glycerol.
54. The method of claim 52 wherein said sterilization step includes
the step of contacting said lens core with ethylene oxide gas.
55. The method of claim 42 wherein said devitalization step
comprises removing epithelium from said anterior surface and
keratocytes from said lens core.
56. The method of claim 42 wherein said revitalization step
comprises adding epithelium to at least a portion of said anterior
surface and keratocytes to said lens core.
57. The method of claim 42 wherein said revitalization step
includes the step of placing said lens core on a polyelectrolyte
gel.
58. The method of claim 42 wherein the corneal tissue matrix of
said lens core is not altered.
Description
FIELD OF THE INVENTION
[0001] This invention is in the field of ophthalmology. More
particularly, it relates to a living contact lens made of donor
corneal tissue, to a method of preparing that lens, and to a
technique of placing the lens on the eye.
BACKGROUND OF THE INVENTION
[0002] The visual system allows the eye to focus light rays into
meaningful images. The most common problem an ophthalmologist or
optometrist will encounter is that of spherical ammetropia, or the
formation of an image by the eye which is out of focus with
accommodation due to an improperly shaped globe. The
ophthalmologist or optometrist determines the refractive status of
the eye and corrects the optical error with contact lenses or
glasses.
[0003] Many procedures have been developed to correct spherical
ammetropia by modifying the shape of the cornea. Light entering the
eye is first focused by the cornea, which possesses approximately
75% of the eye's overall refractory power. The majority of
refractive operations involve either decreasing or increasing the
anterior curvature of the cornea.
[0004] The procedures in early corneal refractive surgery such as
keratophakia and keratomileusis were originally developed to
correct myopia and involved removing a corneal disc from the
patient with a microkeratome. The removed corneal disc was then
frozen prior to reshaping the posterior surface with a cryolathe.
After thawing, the disc was returned to the eye and secured with
sutures.
[0005] Epikeratophakia, as described in U.S. Pat. No. 4,662,881, is
a procedure that involves inserting a precut donor corneal tissue
lens with bevelled edges into corresponding grooves in recipient
cornea. The lens is then sutured to the corneal bed. The donor lens
is lyophilized and requires rehydration before placement on
recipient cornea.
[0006] These techniques and their variations were generally
considered to be unsuccessful due to frequent complications
involving irregular astigmatism, delayed surgical healing, corneal
scarring, and instability of the refractive result. The problems
were attributed to the technical complexity of the procedures as
well as to the distortion in architecture of the corneal tissue
secondary to lens manipulation. For example, in epikeratophakia,
epithelial irregularity is induced by lyophilization of the donor
lens. Freezing of the lenticule in keratophakia and keratomileusis
also causes severe damage to epithelial and stromal cells and
disrupts the lamellar architecture of the cornea.
[0007] The present invention is a pre-fabricated lens made of donor
corneal tissue obtained from tissue sources such as human or animal
cornea. The lens is a corneal disc that is preferably shaped on the
posterior surface generally to conform in shape to the eye's
anterior surface. The inventive lens may be shaped by an ablative
laser, e.g., by an excimer laser or other suitable laser. The
corneal lenticule is living tissue that has not been frozen,
lyophilized, or chemically modified, e.g., fixed with
glutaraldehyde to crosslink corneal tissue. Pre-existing
keratocytes are removed and then replaced with human keratocytes to
decrease antigenicity. After removal of epithelium in the central
zone of the recipient's cornea, the lens is placed on this zone in
the same manner that a contact lens is placed on the eye.
[0008] Ocular lenses found in the prior art do not use native
cornea, but are formulated using soluble collagen such as collagen
hydrogels, e.g., polyhydroxyethylmethacrylate, or other
biocompatible materials. For example, in U.S. Pat. No. 5,213,720,
to Civerchia, soluble collagen is gelled and crosslinked to produce
an artificial lens. In addition to hydrogels, U.S. Pat. No.
4,715,858, to Lindstrom, discloses lenses made from various
polymers, silicone, and cellulose acetate butyrate.
[0009] In the cases where ocular lenses use corneal tissue, the
lenses are either corneal implants or require a separate agent to
adhere the lens to the corneal bed. U.S. Pat. No. 5,171,318, to
Gibson et al., and U.S. Pat. No. 5,919,185, to Peyman, relate to a
disc of corneal tissue that is partially or entirely embedded in
stroma. The ocular lens device disclosed in U.S. Pat. No.
4,646,720, to Peyman et al., and U.S. Pat. No. 5,192,316, to Ting,
is attached to recipient cornea with sutures. The corneal inlay
described in U.S. Pat. No. 4,676,790, to Kern, is bonded to
recipient cornea using sutures, laser welding, or application of a
liquid adhesive or crosslinking solution.
[0010] The ocular lens device of this invention does not alter the
anatomical structure of corneal tissue. U.S. Pat. No. 4,346,482, to
Tennant et al., discloses a "living contact lens" consisting of
donor cornea that has been anteriorly curved for correction of
vision. However, this lens is frozen prior to reshaping on a lathe
which results in stromal keratocyte death. U.S. Pat. No. 4,793,344,
to Cumming et al., also describes a donor corneal tissue lens that
is modified by treatment with a gluteraldehyde fixative that
preserves the tissue and prevents lens swelling. This treatment
alters the basic structure of the corneal lenticule by crosslinking
the tissue.
[0011] Furthermore, the cited documents do not show any methods of
lens preparation that remove native corneal tissue cells and
replace them with cells cultivated from human cornea. My inventive
device is devitalized of native epithelium and keratocytes to
create an acellular corneal tissue, and then revitalized with human
epithelium and keratocytes. An attempt to construct a so-called
"corneal tissue equivalent" was shown in U.S. Pat. No. 5,374,515,
to Parenteau et al. However, the collagen used in that "equivalent"
is obtained from bovine tendon instead of from cornea. The added
keratocytes and epithelium are also not from human sources. The
tissue using these cell culturing procedures is also quite
fragile.
[0012] An excimer laser is used to reform a cornea via the "laser
in situ keratomileusis" (LASIK) procedure. In this technique, an
excimer laser is used to perform stromal photoablation of a corneal
flap or in situ photoablation of the exposed stromal bed. Studies
have shown that the inaccuracy of correction by this procedure may
be as much as one diopter from the desired value. Lenses (contacts
and spectacles), in contrast, are able to correct within 0.25
diopters of the desired value.
[0013] U.S. Pat. No. 6,036,683, to Jean et al., shows the use of a
laser to reshape the cornea. However, the laser changes the native
structure of the cornea by irreversibly coagulating collagen.
Post-laser relaxation of collagen is not possible with this
treatment.
[0014] This invention is a pre-fabricated donor contact lens that
adheres to recipient cornea without sutures. The lens preserves the
anatomy of normal corneal tissue. The donor lens can be obtained
from human and animal sources, is devitalized of native keratocytes
and epithelium to create an acellular tissue, and then revitalized
with human keratocytes and epithelium to maintain lens viability
and decrease antigenicity. The inventive corneal overlay technique
may be completed under local anesthesia as well as general
anesthesia, and the availability of a precut lens will greatly
decrease procedure time, patient cost, and risk of operative
complications. The duration of healing will also be reduced due to
the implementation of a lens already repopulated with
keratocytes.
SUMMARY OF THE INVENTION
[0015] This invention is a pre-fabricated ocular contact lens
device having a lens core made of donor corneal tissue from tissue
sources such as human or animal cornea. The device has a generally
convex anterior surface and a concave posterior surface. The stroma
portion of the lens core may be repopulated with replaced
keratocytes and the anterior surface is preferably covered with a
replaced epithelium. The lens core adheres to recipient cornea
without sutures.
[0016] The lens core may be variously used to correct astigmatism,
myopia, aphakia, and presbyopia. The lens core may be made of
transgenic or xenogenic corneal tissue and have a clarity at least
85% of that of human corneal tissue of a corresponding thickness.
The lens core is not frozen, lyophilized, or chemically treated
with a fixative. However, variations of the device may contain
therapeutic agents, growth factors, or immunosuppressive
agents.
[0017] Another component of the invention is a method for preparing
the lens device. After sharp dissection of a lenticule from donor
corneal tissue, the posterior surface is shaped using an ablative
laser, such as an excimer laser or other suitable shaping lasers.
Native epithelium and keratocytes are removed and then replaced
with human epithelium and keratocytes.
[0018] Another portion of the invention is a method of corneal
overlay that involves de-epithelialization of a portion of the
anterior surface of the recipient cornea and placement of the
inventive ocular lens device upon that anterior surface.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 is a superior, cross-sectional view of the eye.
[0020] FIG. 2A is a side view of the focusing point in myopia.
[0021] FIG. 2B is a side view of a focusing point corrected by
flattening the anterior curvature of the cornea.
[0022] FIG. 3A is a side, cross-sectional view of a pre-fabricated
donor lens.
[0023] FIG. 3B is a side, cross-sectional view of a pre-fabricated
donor lens suitable for correcting myopia.
[0024] FIG. 3C is a side, cross-sectional view of a pre-fabricated
donor lens suitable for correcting aphakia.
[0025] FIG. 3D is a front view of a pre-fabricated donor lens
suitable for bifocal use.
[0026] FIG. 3E is a side, cross-sectional view of the FIG. 3C lens
positioned away from the cornea of an eye.
[0027] FIG. 4A is a side, cross-sectional view of an area of
de-epithelialized recipient cornea prepared to receive the optical
lens of the present invention.
[0028] FIG. 4B is a side, cross-sectional view of the donor lens
after placement on recipient cornea.
DETAILED DESCRIPTION
[0029] The eye is designed to focus light onto specialized
receptors in the retina that turn quanta of light energy into nerve
action potentials. As shown in FIG. 1, light rays are first
transmitted through the cornea (100) of the eye. The cornea is
transparent due to the highly organized structure of its collagen
fibrils. The margins of the cornea merge with a tough
fibrocollagenous sclera (102) and is referred to as the
corneo-scleral layer.
[0030] The cornea (100) is the portion of the corneo-scleral layer
enclosing the anterior one-sixth of the eye. The smooth curvature
of the cornea is the major focusing power of images on the retina
(104) and it provides much of the eye's 60 diopters of converging
power. The cornea is an avascular structure and is sustained by
diffusion of nutrients and oxygen from the aqueous humor (106).
Some oxygen is also derived from the external environment. The
avascular nature of the cornea decreases the immunogenicity of the
tissue, increasing the success rate of corneal transplants.
[0031] The cornea consists of five layers. The outer surface is
lined by stratified squamous epithelium which is about 5 cells
thick. Failure of epithelialization results in necrosis of the
stromal cap and potential scarring of recipient cornea. The
epithelium is supported by a specialized basement membrane known as
Bowman's membrane, which gives the cornea a smooth optical surface.
The bulk of the cornea, the substantia propria (stroma), consists
of a highly regular form of dense collagenous connective tissue
forming thin lamellae. Between the lamellae are spindle-shaped
keratocytes which can be stimulated to synthesize components of the
connective tissue. The inner surface of the cornea is lined by a
layer of flattened endothelial cells which are supported by
Descemet's membrane, a very thick elastic basement membrane.
[0032] As previously mentioned, the focusing power of the cornea is
primarily dependent on the radius of curvature of its external
surface. In myopia, as seen in FIG. 2A, increased curvature of the
cornea (200) causes the focusing point of light rays (202) to fall
short of the retina (204). In FIG. 2B, flattening the anterior
curvature of the cornea (206) corrects the focal point (208).
[0033] Inventive Lens Structure
[0034] The inventive contact lens is of a size and configuration
that upon installation on the cornea, supplements the curvature of
the cornea to correct conditions such as astigmatism, myopia,
hyperopia, presbyopia, and aphakia. The lens core may comprise or
consist essentially of acellular donor corneal tissue which has
been revitalized and then placed on a de-epithelialized host
cornea; the lens core is formed to correct refraction. The donor
lenticule or lens core may be obtained from other human
(allogeneic) or foreign tissue (xenogenic) sources, such as from
rabbit, bovine, porcine, or guinea pig corneal tissue. Ocular
lenses may also come from transgenic corneal tissue or corneal
tissue grown in vitro. In all instances, the architecture of the
corneal layers, the normal corneal tissue matrix, e.g., the
connective tissue or the stroma, is preserved. The "corneal tissue
matrix" is made up of thin layers of collagen fibrils. By the term
"donor corneal tissue", as used here, is meant donor or harvested
corneas or corneal tissue containing the "corneal tissue matrix".
Additionally, it is highly desirable to preserve the anterior
surface of the donated corneal tissue as found beneath the native
epithelium. The donor corneal tissue is not to undergo treatments
such as lyophilization, freezing, or other chemical fixation.
[0035] The ocular lens device of this invention desirably includes
Bowman's membrane, where the donor tissue includes it, to maintain
the native structure of human epithelium. Again, it is highly
desirable to harvest from donor sources in such a way that the
native anterior surface below the epithelium is preserved. I have
found that these native structures have a superior ability, after
the revitalization steps discussed below, to support and maintain
the replaced epithelium also discussed below. Clarity of the
inventive tissue lens core will be at least 85%, preferably between
75%-100%, and most preferably at least 90% of that of human corneal
tissue of corresponding thickness.
[0036] The overall diameter of the inventive lens is generally less
than about 25 mm and more preferably in between 10 and 15 mm. The
thickness of the resulting lens is generally less than 300 .mu.m,
more preferably between 5-100 .mu.m.
[0037] As shown in FIG. 3B, a lens core (316) for myopic patients
is formed, preferably using the procedures discussed below, in such
a way that a generally circular region (318) in the center flattens
its anterior curvature. In correction of aphakia, a lens such as is
shown in FIG. 3C is formed having a comparatively thicked center
(322) and a thinner perimeter (324). In general, the shapes
discussed here are similar to those found in the so-called "soft"
contact lenses and instruction may be had from that technology
relating to the overall form of the lenses selected for correcting
specific ocular maladies.
[0038] As shown in FIGS. 3D and 3E, the inventive lens may also be
used to correct presbyopia. In particular, to treat presbyopia, the
lens (330) is also provided with an generally opaque annular region
(332) in the center. The open center (334) preferably has
plano-lens characteristics and an effective diameter of less than
about 1.5 mm, preferably between about 0.5-1.5 mm, and most
preferably between 0.75 mm and 1.75 mm. The diameter of the central
area or "pinhole" is generally formed to be less than the pupillary
diameter of the eye in daylight. This creates a "pin-hole" effect,
thereby lengthening the overall focal length of the eye and
minimizing the need for the eye to accommodate. Other bifocal lens
designs can also be incorporated, e.g., concentric, segmented, or
progressive diffractive.
[0039] FIG. 3E shows a side, cross-sectional view of the inventive
lens (330) adjacent the anterior surface of a cornea (344) to
illustrate certain features of this variation. The outer diameter
(336) of the opaque annular ring (332) is generally selected so
that it is smaller than the diameter (338) of the pupil (340) in
the iris (342) in low light conditions. In this way, the eye's
cornea and lens and the inventive lens cooperate in such a way that
incident light passes both though the center of the opaque ring
(334), but more importantly, around the periphery of the opaque
ring (332), to allow corrected sight during low light
conditions.
[0040] The annular ring may be situated on the lens core either by
placement of a suitable dye, i.e., by "tattooing", or by placement
of an opaque biocompatible member of, e.g., Dacron mesh or the
like, on the posterior surface to filter light rays.
[0041] Shaping Step
[0042] Returning to FIG. 3A, the donor ocular lens (300) desirably
is obtained after slicing corneal tissue from the donor with a
microkeratome to form a lens core. The donor lens has a structural
surface, the anterior surface of the lens core being the structural
surface of the donor corneal tissue. The lens core anterior surface
is harvested preferably to retain the Bowman's membrane (where the
donor lens contains one) and epithelium (302). The posterior
surface (304) of the resulting inventive lens is generally concave
in shape. It is made so by a shaping step which preferably involves
the use of an ablative laser, such as an excimer laser, to obtain
the necessary power of the lens. Another suitable forming step is
high pressure water jet cutting.
[0043] Sterilization, Devitalization, and Revitalization Steps
[0044] Although the order of the process steps outlined below is
typical, it should be understood that such steps may be varied as
needed to produce the desired result.
[0045] Generally, the lens will first be shaped to an appropriate
shape as discussed above. The lens core may then be subjected to
processes of sterilization, devitalization, and revitalization.
Removal of epithelium (de-epithelialization) and keratocytes
(acellularization) from the donor lens will be referred to as
"devitalization". The addition of human epithelium and keratocytes
will be referred to as "revitalization". One desirable method for
accomplishing those steps is found just below. Other methods are
known.
[0046] Phosphate buffered saline (PBS) with antibiotics, epithelial
cell media, and keratocyte media are solutions used during these
processes. The "PBS with antibiotics" solution may contain:
[0047] PBS with antibiotics
[0048] 1. Amphotericin B (ICN Biomedicals) 0.625 .mu.g/ml
[0049] 2. Penicillin (Gibco BRL) 100 IU/ml
[0050] 3. Streptomycin (Gibco BRL) 100 .mu.g/ml
[0051] 4. Phosphate buffered saline (Gibco BRL)
[0052] The composition of the epithelial cell media may
include:
[0053] Epithelial cell media
[0054] 1. Dulbecco's Modified Eagle Media/Ham's F12 media (Gibco
BRL) 3:1
[0055] 2. 10% fetal calf serum (Gibco BRL)
[0056] 3. Epidermal growth factor (ICN Biomedicals) 10 ng/ml
[0057] 4. Hydrocortisone (Sigma-Aldrich) 0.4 .mu.g/ml
[0058] 5. Cholera toxin (ICN Biomedicals) 10.sup.-10 M
[0059] 6. Adenine (Sigma-Aldrich) 1.8.times.10.sup.-4 M
[0060] 7. Insulin (ICN Biomedicals) 5 .mu.g/ml
[0061] 8. Transferrin (ICN Biomedicals) 5 .mu.g/ml
[0062] 9. Glutamine (Sigma-Aldrich) 2.times.10.sup.-3 M
[0063] 10. Triiodothyronine (ICN Biomedicals) 2.times.10.sup.-7
M
[0064] 11. Amphotericin B (ICN Biomedicals) 0.625 .mu.g/ml
[0065] 12. Penicillin (Gibco BRL) 100 IU/ml
[0066] 13. Streptomycin (Gibco BRL) 100 .mu.g/ml
[0067] The composition of the keratocyte media may include:
[0068] Keratocyte media
[0069] 1. DMEM
[0070] 2. 10% neonatal calf serum (Gibco BRL)
[0071] 3. Glutamine (Sigma-Aldrich) 2.times.10.sup.-3 M
[0072] 4. Amphotericin B (ICN Biomedicals) 0.625 .mu.g/ml
[0073] Sterilization Step
[0074] After harvesting the lens core from donor corneal tissue and
following the shaping step, the lens may be sterilized by immersion
into 98% glycerol at room temperature. Three weeks of glycerol
treatment inactivates intracellular viruses and any bacteria or
fungi. Ethylene oxide gas sterilization may also be used, but tends
to induce variable damage to stromal tissue.
[0075] Devitalization Step
[0076] De-Epithelialization
[0077] Following sterilization, I prefer to de-epithelialize the
donor lens by placing it in sterile PBS with antibiotics for four
hours and changing the solution many times. The lens core may then
be kept submerged in the PBS solution at 37.degree. C. for one week
to produce a split between the epithelium and the stroma. The
epithelium may then be removed, e.g., by physically scraping or
washing with a liquid stream. Small numbers of lenses may be
stripped of epithelium by gentle scraping with forceps.
[0078] Acellularization
[0079] The de-epithelialized lens may then be immersed in sterile
PBS with antibiotics for an appropriate period, e.g., several
weeks, perhaps six weeks to remove native keratocytes. The solution
may be changed twice weekly. In some instances, it may not be
necessary to remove keratocytes from the donor lens, e.g., when the
donor tissue is obtained from a transgenic source and has minimal
antigenicity.
[0080] Revitalization Step
[0081] Preparation of Cells
[0082] Human epithelial cells and keratocytes are used in the
revitalization process. Epithelial cells may be obtained from a
tissue bank, but are preferably obtained from fetal or neonatal
tissue. Fetal cells are most preferable, since the properties of
fetal tissue minimize scarring during the wound healing
process.
[0083] In any event, freshly isolated epithelial cells, obtained by
trypsinization of corneal tissue, may be seeded onto a precoated
feeder layer of lethally irradiated 3T3 fibroblasts (i.3T3) in
epithelial cell media. Cells are cultured and media changed every
three days until the cells are 80% confluent, about 7-9 days.
Residual i.3T3 are removed with 0.02% EDTA (Sigma-Aldrich) before
the epithelial cells are detached using trypsin (ICN Biomedicals).
Another method of regenerating epithelium involves culturing
autologous epithelial cells on human amniotic membrane as described
in Tsai et al. (2000). "Reconstruction of Damaged Corneas by
Transplantation of Autologous Limbal Epithelial Cells," New England
Journal of Medicine 343:86-93.
[0084] Keratocytes may be extracted from the remaining stromal
tissue. The stroma is washed in PBS, finely minced, and placed in
0.5% collagenase A (ICN Biomedicals) at 37.degree. C. for 16 hours.
Keratocytes obtained from this enzyme digest are then serially
cultured in keratocyte media. The epithelial cells and keratocytes
generated in the revitalization step will be referred to as
"replaced" epithelium and keratocytes.
[0085] Production of the Donor Lens
[0086] The acellular donor lens core may then be placed on a
hydophilic, polyelectrolyte gel for completion of the
re-vitalization. The preferred polyelectrolytes are chondroitin
sulfate, hyaluronic acid, and polyacrylamide. Most preferred is
polyacrylic acid. The lens is immersed in keratocyte media and
incubated with approximately 3.times.10.sup.5 keratocytes for 48
hours at 37.degree. C. Approximately the same amount of epithelial
cells are then added to the anterior stromal surface. Tissue
culture incubation continues for another 48 hours. Keratocyte media
is changed every two to three days. Once the epithelium is
regenerated, the polyelectrolyte gel draws water out of the lens at
a pressure of about 20-30 mm Hg until the original lens dimensions
are obtained.
[0087] Replaced epithelium covers at least a portion of the
anterior surface and replaced keratocytes repopulate the stroma of
the lens core after revitalization. It may be beneficial in some
instances to incorporate therapeutic agents, growth factors, or
immunosuppressive agents into the lens core to further decrease the
risk of rejection or remedy disease states.
[0088] Placement of the Lens on the Eye
[0089] During the procedure, the donor lens (300) is placed on a
portion of recipient cornea that has been de-epithelialized (308).
The result is the construct (312) shown in FIG. 4B. The lens'
replaced epithelium and the host epithelium eventually grow to form
a continuous, water-tight layer (310). I have found that the
inventive lens bonds to recipient cornea without sutures or
adhesives, but can also be removed without substantial
difficulty.
[0090] I have described the structural and physiologic properties
and benefits of this donor ocular lens. This manner of describing
the invention should not, however, be taken as limiting the scope
of the invention in any way.
* * * * *