U.S. patent application number 10/767408 was filed with the patent office on 2005-02-03 for microfabricated tissue as a substrate for pigment epithelium transplantation.
Invention is credited to Bent, Stacey F., Blumenkranz, Mark, Fishman, Harvey A., Huie, Philip JR., Lee, Christina, Palanker, Daniel V..
Application Number | 20050027356 10/767408 |
Document ID | / |
Family ID | 25359715 |
Filed Date | 2005-02-03 |
United States Patent
Application |
20050027356 |
Kind Code |
A1 |
Fishman, Harvey A. ; et
al. |
February 3, 2005 |
Microfabricated tissue as a substrate for pigment epithelium
transplantation
Abstract
An ocular implant is provided with a substrate and a membranous
tissue layer secured to the substrate. Cells such as IPE cells, RPE
cells and stem cells are attached on the surface of the membranous
tissue layer either in situ or in vivo through cells
transplantation. The cells are separated into regions on the
surface by creating a pattern on the surface enclosing regions for
receiving the cells. The substrate is a bioabsorbable and/or
polymeric substrate. Examples of membranous tissue layer are lens
capsule, inner limiting membrane, corneal tissue, Bruch's membrane
tissue, amniotic membrane tissue, serosal membrane tissue, mucosal
membrane tissue and neurological tissue. The membranous tissue
layer could have a micropattern of biomolecules. A microfluidic
network could be placed onto the microfabricated membranous tissue
layer.
Inventors: |
Fishman, Harvey A.; (Menlo
Park, CA) ; Blumenkranz, Mark; (Portola Valley,
CA) ; Bent, Stacey F.; (Palo Alto, CA) ; Lee,
Christina; (San Francisco, CA) ; Huie, Philip
JR.; (Cupertino, CA) ; Palanker, Daniel V.;
(Sunnyvale, CA) |
Correspondence
Address: |
LUMEN INTELLECTUAL PROPERTY SERVICES, INC.
2345 YALE STREET, 2ND FLOOR
PALO ALTO
CA
94306
US
|
Family ID: |
25359715 |
Appl. No.: |
10/767408 |
Filed: |
January 28, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10767408 |
Jan 28, 2004 |
|
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|
09872513 |
Jun 1, 2001 |
|
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|
Current U.S.
Class: |
623/6.63 ;
623/23.72; 623/4.1 |
Current CPC
Class: |
A61L 27/3604 20130101;
A61F 2/14 20130101; A61L 27/3839 20130101; A61L 27/3813
20130101 |
Class at
Publication: |
623/006.63 ;
623/004.1; 623/023.72 |
International
Class: |
A61F 002/14; A61F
002/02 |
Claims
What is claimed is:
1. An ocular implant, comprising: (a) a bioabsorbable substrate;
(b) a microfabricated membranous tissue layer secured to said
bioabsorbable substrate; and (c) cells on the surface of said
microfabricated membranous tissue layer, said cells separated into
regions on said surface by creating a pattern on said surface
enclosing said regions for receiving said cells.
2. The ocular implant as set forth in claim 1, wherein said tissue
of said microfabricated membranous tissue layer is selected from
the group consisting of lens capsule, inner limiting membrane,
comeal tissue, Bruch's membrane tissue, amniotic membrane tissue,
serosal membrane tissue, mucosal membrane tissue and neurological
tissue.
3. The ocular implant as set forth in claim 1, wherein said cells
are cells selected from the group consisting of IPE cells, RPE
cells and stem cells.
4. The ocular implant as set forth in claim 1, wherein said cells
are received on said surface in situ or in vivo.
5. The ocular implant as set forth in claim 1, wherein said cells
on said microfabricated membranous tissue layer are separated by
growth inhibitory barriers.
6. The ocular implant as set forth in claim 1, wherein said cells
are separated by a patterned stencil.
7. The ocular implant as set forth in claim 1, wherein said
bioabsorbable substrate comprises a material selected from the
group consisting of glass, collagen, glycosaminoglycans, chitosan;
poly(hydroxyalkanoates), poly(.alpha.-hydroxy acids), polyglycolic
acid (PGA), polylactic acid (PLA), polylactide-polyglycolide
(PGA-PLA) mixtures, alloys and copolymers (PLGA), poly(dioxanones),
poly(E-caprolactone); poly(ortho esters), poly(anhydrides),
poly(phosphazenes), poly(amino acids), and other compounds,
polymers, copolymers, alloys, mixtures and combinations
thereof.
8. The ocular implant as set forth in claim 1, wherein said
bioabsorbable substrate is formed of a material selected from the
group consisting of poly-lactic acid, polyglycolic acid,
polyorthoesters, polyanhydrides, polyphosphazines, poly-lactic acid
glycolic acid copolymers, polyethylene glycol/polylactic acid
copolymers and blends and copolymers thereof.
9. The ocular implant as set forth in claim 1, wherein said
microfabricated membranous tissue layer is about 2 to about 5
micrometers in thickness.
10. The ocular implant as set forth in claim 1, wherein said
microfabricated membranous tissue layer has micropores or pits.
11. The ocular implant as set forth in claim 1, wherein said
microfabricated membranous tissue layer has a micropattern of
biomolecules.
12. The ocular implant as set forth in claim 11, wherein said
biomolecules of said micropattern of said microfabricated
membranous tissue layer are selected from the group consisting of
proteins, peptides, organic molecules, oligosaccharides, and small
chain polymers.
13. The ocular implant as set forth in claim 11, wherein one or
more of said biomolecules of said micropattern of said
microfabricated membranous tissue layer are selected from the group
consisting of poly (methyl methacrylate), polystyrene, poly (methyl
styrene), collagen, keratin sulfate, hyaluronic acid,
glycosaminoglycan, octadecyltrichlorosilane, silane polymers,
polylysine, polylactic glycolic acid (PLGA)-derivatized polylysine
and polylysine peptides.
14. The ocular implant as set forth in claim 1, wherein said ocular
implant is in a subretinal space.
15. The ocular implant as set forth in claim 1, further comprising
a microfluidic network placed onto said microfabricated membranous
tissue layer.
16. An implant, comprising: (a) a polymeric substrate; (b) a
membranous tissue layer secured to said polymeric substrate; and
(c) an array of cells on the surface of said membranous tissue
layer, wherein said cells are separated into regions on said
surface defining said array by creating a pattern on said surface
enclosing said regions for receiving said cells.
17. The implant as set forth in claim 16, wherein said tissue of
said microfabricated membranous tissue layer is selected from the
group consisting of lens capsule, inner limiting membrane, corneal
tissue, Bruch's membrane tissue, amniotic membrane tissue, serosal
membrane tissue, mucosal membrane tissue and neurological
tissue.
18. The implant as set forth in claim 16, wherein said cells are
cells selected from the group consisting of IPE cells, RPE cells
and stem cells.
19. The implant as set forth in claim 16, wherein said cells are
received on said surface in situ or in vivo.
20. The implant as set forth in claim 16, wherein said cells on
said microfabricated membranous tissue layer are separated by
growth inhibitory barriers.
21. The implant as set forth in claim 16, wherein said cells on
said membranous tissue layer are in a predetermined pattern as
depicted in FIG. 3 or FIG. 5.
22. The implant as set forth in claim 16, wherein said cells are
separated by a patterned stencil.
23. The implant as set forth in claim 16, wherein said
bioabsorbable substrate comprises a material selected from the
group consisting of glass, collagen, glycosaminoglycans, chitosan;
poly(hydroxyalkanoates), poly(.alpha.-hydroxy acids), polyglycolic
acid (PGA), polylactic acid (PLA), polylactide-polyglycolide
(PGA-PLA) mixtures, alloys and copolymers (PLGA), poly(dioxanones),
poly(E-caprolactone); poly(ortho esters), poly(anhydrides),
poly(phosphazenes), poly(amino acids), and other compounds,
polymers, copolymers, alloys, mixtures and combinations
thereof.
24. The implant as set forth in claim 16, wherein said polymeric
substrate is formed of a material selected from the group
consisting of poly-lactic acid, polyglycolic acid, polyorthoesters,
polyanhydrides, polyphosphazines, poly-lactic acid glycolic acid
copolymers, polyethylene glycol/polylactic acid copolymers and
blends and copolymers thereof.
25. The implant as set forth in claim 16, wherein said membranous
tissue layer is about 2 to about 5 micrometers in thickness.
26. The implant as set forth in claim 16, wherein said membranous
tissue layer has micropores or pits.
27. The implant as set forth in claim 16, wherein said membranous
tissue layer has a micropattern of biomolecules.
28. The implant as set forth in claim 27, wherein said biomolecules
of said micropattern of said membranous tissue layer are selected
from the group consisting of proteins, peptides, organic molecules,
oligosaccharides, and small chain polymers.
29. The implant as set forth in claim 27, wherein one or more of
said biomolecules of said micropattern of said membranous tissue
layer are selected from the group consisting of poly (methyl
methacrylate), polystyrene, poly (methyl styrene), collagen,
keratin sulfate, hyaluronic acid, glycosaminoglycan,
octadecyltrichlorosilane, silane polymers, polylysine, polylactic
glycolic acid (PLGA)-derivatized polylysine and polylysine
peptides.
30. The implant as set forth in claim 16, wherein said implant is
in a subretinal space.
31. The implant as set forth in claim 16, further comprising a
microfluidic network placed onto said microfabricated membranous
tissue layer.
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to the field of
treatment of eye disorders, in particular retinal disorders such as
age-related macular degeneration, retinitis pigmentosa, and other
retinal diseases. In addition, the invention relates to methods and
apparatus for modifying tissues, and for the transplantation of
cells and tissues.
BACKGROUND OF THE INVENTION
[0002] Diseases of the retina, such as age-related macular
degeneration (AMD), retinitis pigmentosa (RP), and other diseases,
are the leading cause of severe visual impairment or blindness in
the industrialized world. One hallmark of AMD, as in RP, is the
degeneration and loss of cells of the retinal pigment epithelium
(RPE). Bruch's membrane is also thought to be damaged; such damage
may be the initiating stimulus for RPE demise. RPE cells are vital
to the survival and proper functioning of retinal photoreceptors,
which are the only cells in the eye which directly sense light. RPE
degeneration in retinal diseases such as AMD and RP is related to
the loss of photoreceptor function and the visual impairment that
is associated with these diseases.
[0003] The RPE is located adjacent to the neural retina, directly
opposed to the retinal photoreceptors. RPE cells in vivo form one
cell thick cobblestone-like tissue linked together by tight
junctions, with differentiated apical and basal membranes. The RPE
cells in vivo grow tightly packed together at high density to form
a tight epithelium that acts as a barrier regulating transport
between the photoreceptors and the underlying Bruch's membrane,
choroid and the choroidal vasculature. The apical portion of the
RPE is adapted to surround and engulf photoreceptor outer segments,
to perform its vital functions of phagocytosis and digestion of
shed photoreceptor tips, and of recycling retinal for re-use in
photopigments. The basal portion of the RPE is apposed to Bruch's
membrane, a highly vascularized supporting membrane which supplies
the RPE and photoreceptors with needed oxygen and nutrients, and
prevents the accumulation of carbon dioxide and other waste
products which would otherwise impair retinal function. Damage to
Bruch's membrane, which may occur due to accumulation of waste
products from outer segment metabolism, for example, prevents the
exchange of oxygen, growth factors and waste products. Such
impaired exchange leads to hypoxia in the photoreceptors. In
response, it is thought that survival signals are sent out to
initiate the in-growth of neovascular vessels, and so to the wet
form of AMD.
[0004] The iris pigment epithelium (IPE), which, like the RPE is
derived from the neuroectoderm of the embryo, is located adjacent
to the iris at the part of the eye opposite to the retina. Thus, in
place in the intact eye, IPE cells are remote from retinal
photoreceptors. Although much about IPE cell physiology and
function remains unknown, like RPE cells, IPE cells in culture have
been shown to be capable of phagocytosis of photoreceptor outer
segments. RPE cells may be grown on artificial substrates (Pfeffer,
B. A., Chapter 10, "Improved Methodology for Cell Culture of Human
and Monkey Retinal Pigment Epithelium," Progress in Retinal
Research, Vol. 10 (1991) Ed. Osborn, N., and Chader, J.; Lu et al.,
J. Biomater. Sci. Polymer Edn. 9:1187-1205 (1998), and Lu et al.,
Biomaterials 20:2351-2361 (1999). In addition, there have been
attempts to use lens capsule tissue as a substrate for growing RPE
and IPE cells (Hartman et al., Graefe's Archiv Clin Exp Ophthalmol
237:940-945 (1999); Nicolini et al., Acta Ophthalmol Scand 2000
October;78(5):527-31)).
[0005] Many approaches have been tried in the treatment of
degenerative and progressive retinal diseases. For example,
attempted treatments for AMD include photodynamic therapy,
radiation therapy, and macular relocation in order to repair,
retard the progression, or compensate for the effects of the
disease. However, such approaches have not met with great
success.
[0006] Since RPE cell loss occurs in many retinal diseases, the
transplantation of cells has great attraction as a therapy and
possible cure for AMD and other diseases. Direct transplantation of
RPE cells into the retina has been attempted in order to replace
lost RPE cells. However, this approach has not succeeded in the
past, due in part to the failure of the transplanted cells to
function properly and in part due to rejection of the cells by the
host animals.
[0007] Transplantation of RPE cells has been suggested as a therapy
for retinal dystrophy (U.S. Pat. No. 5,962,027 to Hughes and U.S.
Pat. No. 6,045,791 to Liu). All patents and publications named
herein, both supra and infra, are hereby incorporated by reference
in their entirety. In addition, experimental evidence that IPE
cells could substitute for RPE cells has led to preliminary
attempts to transplant IPE cells in animals and in order to
ameliorate symptoms of AMD (Abe et al., Tohoku J. Exp. Med.
189:295-305 (1999), Abe et al., Cell Transplantation 8(5):501-10
(1999); Schraermeyer et al., Invest. Opth. Vis. Sci. 40(7):1545-56
(1999); Thumann et al., Transplantation 68(2)195-201 (1999); Abe et
al., Tohoku J. Exp. Med. 191:7-20 (2000); Abe et al., Current Eye
Research 20(4):268-275 (2000); Lappas et al., Graefes 's Arch Clin
Exp Ophthalmol 238:631-641 (2000), Thumann, et al., Arch.
Ophthalmol. 118:1350-1355 (2000)).
[0008] However, challenges to both IPE and RPE transplantation
methods include i) difficulty in repairing the diseased Bruch's
membrane, ii) inability to secure and position newly transplanted
cells, and iii) lack of control over extracellular matrix signaling
molecules that are critical to the structure, function, and
survival of the pigment epithelial cell. For these and other
reasons, techniques for IPE and RPE transplantation using
antibiotics or immunosuppressants have not been successful. There
has been no demonstration of significant visual improvement with
these approaches, and problems of tissue reintegration remain.
Thus, despite the apparent promise of the transplantation approach,
AMD and other retinal diseases remain without successful
therapeutic interventions.
[0009] Accordingly, there is need in the art for novel methods and
apparatus for modification of tissues for transplantation and for
transplantation of such tissues for the relief of retinal
diseases.
BRIEF SUMMARY OF THE INVENTION
[0010] The present invention is directed to methods, apparatus, and
related products for modifying tissues and growing cells for
transplantation. In particular, the invention is directed towards
methods, apparatus, and related products for transplantation of
cells and tissues into the retina for treatment of retinal diseases
such as AMD and RP. Tissues modified by the methods disclosed
herein are termed microfabricated tissues. The invention includes
microfabricated membranous tissues, including microfabricated
ocular membranous tissues, for example microfabricated lens capsule
tissues, microfabricated inner limiting membrane tissues,
microfabricated Bruch's membrane tissues, and other tissues. The
invention further includes microfabricated membranous tissues for
use in transplantation, methods for microfabricating membranous
tissues, methods for using microfabricated membranous tissues,
methods for growing cells on microfabricated membranous tissues,
and methods for transplanting microfabricated tissues and cells
into the eye of an animal. For example, the animal may be a
human.
[0011] A microfabricated membranous tissue embodying features of
the invention may be prepared by contacting membranous tissue with
a substrate including a bioabsorbable material, which may be
submersed in phosphate buffered saline, or by coating a surface of
a membranous tissue with a bioabsorbable material, and modifying
the membranous tissue either before or after coating or contacting
the tissue with the substrate. Suitable bioabsorbable materials
include collagen; glycosaminoglycans; chitosan;
poly(hydroxyalkanoates); poly(.alpha.-hydroxy acids); polyglycolic
acid (PGA); polylactic acid (PLA); polylactide-polyglycolide
(PGA-PLA) mixtures, alloys and copolymers (PLGA); poly(dioxanones);
poly(E-caprolactone); poly(ortho esters); poly(anhydrides);
poly(phosphazenes); poly(amino acids); and other compounds,
polymers, copolymers, alloys, mixtures and combinations of these
compounds. Suitable membranous tissue includes lens capsule, inner
limiting membrane, Bruch's membrane, corneal tissue, amniotic
membrane, serosal membrane tissue, mucosal membrane tissue, and
other tissue including neurological tissue.
[0012] A microfabricated membranous tissue, coated with, in contact
with, or placed on a substrate, may further have cells grown upon
it, by a method including coating membranous tissue or contacting
membranous tissue with a substrate, the tissue optionally being
submersed in phosphate buffered saline or other physiological
solution, modifying the membranous tissue, and applying cells (such
as IPE and RPE cells) to the modified membranous tissue. A
microfabricated membranous tissue may also be modified by partly
covering the membranous tissue with a stencil and growing cells on
the exposed surface of the membranous tissue.
[0013] Methods for modifying membranous tissues may include
mechanical methods including mechanical ablation, mechanical
contact, and photoablation methods. The methods of the invention
for modifying membranous tissues may be applied to a variety of
tissues, including ocular membranous tissues. For example, the
methods of the invention include methods for modifying lens capsule
tissue, such as human lens capsule tissue, and for modifying inner
limiting membrane tissue, such as human inner limiting membrane
tissue.
[0014] Methods for modifying membranous tissues include bulk
modification methods and surface modification methods. Surface
modification methods and bulk modification methods may be applied
alone, or may each be applied together to the same membranous
tissue. Modification of the surface and bulk properties of the
membranous tissue improves the tissue's suitability for
transplantation into an animal. Such tissue modification may
improve the ability of cells to attach and grow on the tissue, and
may improve the permeability properties of the tissue so that
nutrients, electrolytes, and other desired substances are better
able to pass through the modified tissue.
[0015] The methods of the invention, whether bulk or surface
modification methods, include removal of membranous tissue, such as
a lens capsule or an inner limiting membrane, from an eye,
flattening the membranous tissue onto a glass or plastic substrate,
such as a coverslip, submersed in phosphate buffered saline, or
flattening the membranous tissue onto a temporary dissolvable
polymer for ease of surgical transplantation. The modified tissue
provides a suitable substrate for cells, and may be exposed to
cells which may attach and grow. The modified tissue, with adherent
cells if any were applied to and grown on the tissue and/or with
polymer, if any, may next be transplanted into a desired location
within the body of an animal. Following transplantation, where the
modified tissue has been prepared with a dissolvable polymer, the
polymer will dissolve and be absorbed by the body of the animal
into which the tissue has been transplanted, leaving the
transplanted tissue and cells in place.
[0016] Suitable dissolvable polymers include poly-lactic acid,
polyglycolic acid, polyorthoesters, poly anhydrides,
polyphosphazines, poly-lactic acid glycolic acid copolymers (PLGA),
including PLGA (e.g., a 50:50 mixture of lactic to glycolic acid
copolymer, a 90:10 mixture, or other proportions), poly-lactic acid
polymers (PLLA), polyethylene glycol/polylactic acid copolymer
(PEG/PLA), and blends and co-polymers thereof.
[0017] Bulk modification methods are those where substantial
portions of the membranous tissue, not limited to surface portions
of the tissue, are modified by the method. Surface modification
methods are those where the membranous tissue is modified at and
near to the surface, but is not greatly modified in other portions
of the tissue.
[0018] Bulk modification methods for modifying membranous tissue,
including ocular membranous tissue such as lens capsule tissue and
inner limiting membrane tissue, include methods for modifying the
thickness, permeability, and other properties of the tissue. Bulk
modification methods include mechanical ablation, including
rubbing, scraping, cutting, and applying tension, contacting the
membranous tissue with a contacting surface such as a stamp, and
producing a micropattern in the membranous tissue. In one
embodiment of the bulk modification method, treatment after removal
and flattening of the membranous tissue includes use of a laser or
ion beam to modify the surface of the membranous tissue to reduce
the overall thickness of the tissue. For example, the lens capsule,
which can normally be up to about 8 to 14 micrometers (.mu.m)
thick, may be ablated by photoablation with an excimer laser to be
about 2 to 5 .mu.m thick, so that the overall thickness of the
altered lens capsule mimics the thickness of Bruch's membrane
(about 2 to 4 .mu.m).
[0019] In another embodiment of the bulk modification method, such
further treatment includes photoablation using a laser, such as an
excimer, titanium sapphire, or YAG laser, or ion beam treatment, to
produce micropores or pits in the membranous tissue. The micropores
may be sized on the order of a few micrometers or less in diameter.
A micropattern of micropores or pits produced in the membranous
tissue by such treatment.
[0020] Membranous tissue may be treated by impregnation with a
deactivated collagenase enzyme that is activated by laser light
illumination. For example, very small regions sized less than a
micrometer in diameter of tissue may be activated by illumination
with a 2-photon confocal laser system.
[0021] Enzymes may be deposited onto the membranous tissue
effective to biologically etch the surface and interior of the
membranous tissue effective to provide desired topology and surface
adhesion properties to the tissue. In some embodiments of this
method, the deposition step includes contacting the membranous
tissue with a contacting surface, such as a microcontact printing
stamp, carrying enzymes effective to biologically etch the surface
and interior of the membranous tissue.
[0022] Treatments may include surface modification of the
membranous tissue as well. For example, treatment may include
deposition of patterns of biomolecules onto membranous tissue, and
production of patterns of pores or pits or other surface features
by laser or ion beam treatment. In some embodiments of this method,
the patterns are sized on the order of a few micrometers or less.
In other embodiments of this method, the biomolecules include
peptides and small chain polymers effective to deactivate selective
cell attachment sites on membranous tissue.
[0023] In one embodiment of the surface modification method,
microcontact printing techniques are used to fabricate chemical
micropatterns of biomolecules on the membranous tissue. Membrane
surfaces may also be modified by mechanical ablation methods
including rubbing, scraping and flowing solutions over the
surface.
[0024] In another embodiment of the surface modification method,
the surface of the membranous tissue is masked to cover part, but
not all, of the surface of the tissue, and then irradiated with
ultraviolet (UV) radiation effective to denature the extracellular
matrix (ECM) of the exposed portions of tissue. In order to
activate only certain portions of the surface of the membranous
tissue, a deactivating substance such as polyvinyl alcohol (PVA) or
mucilage can be applied to the surface of the tissue, and an
excimer laser can be used to ablate a micropattern on the
membranous tissue surface through an irradiation mask.
[0025] The masking step may be performed by placing a grid onto the
surface of the membranous tissue, or by using microcontact printing
techniques to apply a pattern of protecting molecules onto the
surface of the membranous tissue effective to prevent ECM
denaturation in regions covered by the protecting molecules or
grid.
[0026] Cells may be grown on microfabricated membranous tissues.
For example, cells may be applied to microfabricated membranous
tissues, either in situ or in vivo through transplantation, which
may have patterns on their surfaces. In further embodiments, the
microfabricated membranous tissue may be lens capsule tissue or
inner limiting membrane tissue, and the cells may be IPE cells. In
yet other embodiments of the invention, the microfabricated
membranous tissues and the cells may be obtained from the same
animal. In this last case, transplantation of the modified tissue
and cells into that animal would be autologous transplantation,
which would not suffer from rejection by the animal's immune
system.
[0027] The invention also provides methods for using
microfabricated membranous tissues, including surgical methods for
transplanting microfabricated membranous tissues into an animal.
The methods for transplanting microfabricated membranous tissues
into an animal include surgical methods for transplanting
microfabricated membranous tissues into the eye of an animal, such
as transplantation of microfabricated lens capsule tissue or
microfabricated inner limiting membrane tissue near to or into the
retina of an animal. The transplanted tissue may further include
cells grown on microfabricated lens capsule tissues or
microfabricated inner limiting membrane tissues. In preferred
embodiments, the transplanted microfabricated membranous tissue
includes IPE cells grown on microfabricated lens capsule tissues or
microfabricated inner limiting membrane tissues, and may be
autologous tissue and cell transplants.
[0028] The invention also provides products useful in fabricating
and using microfabricated tissues. Such products include products
and tools for making modified ocular membranous tissues, including
microfabricated lens capsule and inner limiting membrane tissues,
and products and tools for transplanting the transplanted tissues
and cells into the eye of an animal.
[0029] The present invention is directed to methods and related
products for treating retinal diseases such as AMD, RP, and other
retinal diseases. For example, one therapy for AMD is to transplant
suspensions of either retinal pigment epithelial (RPE) cells, iris
pigment epithelial (IPE) cells, stem cells, or other cells, to
rescue the diseased retina. The present invention provides novel
tissue engineering techniques to precision engineer autologous
human tissues (e.g., human lens capsule) as a substrate for
transplanting cells, such as IPE cells, RPE cells, stem cells, and
other cells. Transplanted pigment epithelium cells grown on the
modified tissue and substrates of the invention are able to grow to
high density and to exhibit features indicative of differentiation,
important characteristics of these cells in normal retinas. In
addition, unlike prior methods, the modified membranous tissues
(including modified ocular membranous tissues, such as lens
capsule, inner limiting membrane, and other substrates provided by
the present invention, and such substrates with growing epithelial
cells) are effective to replace many of the functions of Bruch's
membrane, which may be damaged in degenerative retinal diseases.
Thus the present methods and apparatus for transplantation of
pigment epithelial cells provide transplanted cells which grow to
high density and are able to perform needed physiological functions
lacking in patients with retinal degenerative diseases.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1A is a cross-sectional view of microfabricated
membranous tissue on a dissolvable substrate embodying features of
the invention.
[0031] FIG. 1B is a cross-sectional view of an eye having
microfabricated tissue on a dissolvable substrate implanted in the
subretinal space of its eye.
[0032] FIG. 1C is a detailed cross-sectional view of the
microfabricated tissue, retina and subretinal space of the eye.
[0033] FIG. 2 illustrates a microcontact printing stamp useful for
producing microfabricated membranous tissue embodying features of
the invention.
[0034] FIG. 3 illustrates microfabricated lens capsule tissue after
contact with a microcontact printing stamp embodying features of
the invention.
[0035] FIG. 4 illustrates a poly(dimethylsiloxane) (PDMS) stamp for
micropatterning membranous tissue according to methods embodying
features of the invention.
[0036] FIG. 5 illustrates cells growing on a human lens capsule
micropatterned with the PDMS stamp illustrated in FIG. 4.
[0037] FIG. 6 is a photomicrograph of a microfabricated lens
capsule on a poly-lactide/polyglycolide carrier matrix.
[0038] FIG. 7 is a photomicrograph of a section of rabbit retina
containing a microfabricated lens capsule on a
poly-lactide/polyglycolide carrier matrix one week after
implantation.
DETAILED DESCRIPTION OF THE INVENTION
[0039] The present invention provides methods and apparatus for
modifying tissues and cells for transplantation. The methods of the
invention for modifying tissues may be applied to a variety of
tissues from a variety of organs. The following definitions are
helpful in describing the invention.
[0040] The term "autologous" is used herein to refer to cells or
tissues derived from the same animal as other cells or tissues;
thus, with respect to a tissue, cells are autologous cells when
they are derived from the same animal as the tissue is derived
from; analogously, the tissue is autologous tissue with respect to
the cells when the cells and tissue are derived from the same
animal.
[0041] The term "biomolecule" is used herein to mean a molecule
that has a biological activity. Thus, a biomolecule is one that,
when in contact with a cell or tissue, acts on or affects that cell
or tissue.
[0042] The term "bulk modification" is used herein to mean the
modification of the properties of substantial portions of a tissue,
where such modification is not limited to the surface portions of
the tissue.
[0043] The term "surface modification" is used herein to mean the
modification of the properties of a tissue at and near to the
surface of the tissue.
[0044] The term "membranous tissue" is used herein to mean any
tissue of an animal that forms a sheet or sheath; membranous tissue
commonly encloses or delimits a tissue, or divides an organ or
tissue into separate compartments. "Ocular membranous tissue" is
used herein to mean membranous tissue derived from the eye of an
animal; lens capsule tissue and inner limiting membrane are
examples of ocular membranous tissue, as are corneal membranes,
Bruch's membrane, and other membranous tissues of the eye.
[0045] The term "ablation" is used herein to mean the alteration of
a tissue, not necessarily including the reduction in the size or
the removal of tissue. As used herein, "mechanical ablation" means
alteration, reduction, or removal of tissue by mechanical action,
such as scraping, scoring, contacting with a contacting surface
(such as a stamp), applying tension, or other mechanical method. As
used herein, "photoablation" means irradiation by ultraviolet
light, laser light, or other radiation, such as by light from an
excimer, titanium sapphire, YAG or other laser, effective to alter
the surface or bulk properties of a tissue. "Ion ablation" is used
herein to refer to surface or bulk modification effected by ion
beam treatment of a membranous tissue.
[0046] A "proteolytic enzyme," or a "protease," is a type of
molecule that is effective to at least partially digest (cut into
pieces) a protein or peptide molecule. Examples of proteases and
proteolytic enzymes include, but are not limited to, collagenase,
trypsin, chymotryptsin, dispase, liberase, thermolysin, pepsin, and
papain.
[0047] The term "transplantation" is used herein to mean the
insertion, deposition or positioning of cells or tissues into an
animal. The deposition of cells growing on modified lens capsule
tissue into the subretinal space is an example of
transplantation.
[0048] The term "microcontact printing" is used herein to mean
deposition of desired molecules onto a surface in a pattern with
features sized on the order of several tens of micrometers or
smaller.
[0049] The term "microfabrication" is used herein to mean the
production of modified tissues by surface modification, bulk
modification, or both.
[0050] The term "microfabricated tissue" is used herein to mean a
tissue that has been altered or modified by microfabrication
methods.
[0051] A "contacting surface" is a surface configured for
contacting a second surface and for depositing molecules initially
present on the contacting surface onto the second surface. A
"stamp," "microfabrication stamp," "microcontact printing stamp,"
"microcontact stamp," or "microfabrication printing stamp" is a
contacting surface, and the terms "stamp," "microfabrication
stamp," "microcontact printing stamp," "microcontact stamp," and
"microfabrication printing stamp" are used herein to mean a device
adapted to deposit desired molecules in a pattern with features
sized on the order of several tens of microns or smaller.
[0052] The term "micropattern" is used herein to mean a pattern,
such as an ordered array, design or contour with features sized on
the order of several tens of microns or smaller.
[0053] By "dissolvable polymer" is meant a polymer that is
biodegradable, and that upon introduction into an animal may at
least partially dissociate and disperse into fluids and tissues of
the animal.
[0054] A "laser" may be an excimer laser, a titanium sapphire
laser, an yttrium-aluminum-garnet (YAG), or other laser. A laser is
capable of emitting a powerful beam of coherent light produced by
light amplification within the laser cavity or crystal of the
laser.
[0055] As used herein "excimer laser" means a laser light source
that provides laser light of a wavelength below about 400
nanometers (nm). Excimer lasers may be xenon, krypton, or fluorine
lasers, or, more preferably may be an argon fluoride laser. An
argon fluoride laser provides laser light in the ultraviolet,
typically with a wavelength of about 193 nm, suitable for ablation
of epithelial, connective, and other tissues. For use in tissue
modification, such as tissue ablation, laser light may be pulsed at
between about 1 to 50 Hz with each pulse having a duration of
between about 1 to 200 nanoseconds (ns), preferably between about
10 to 20 ns. Laser beams, such as produced by argon fluoride
lasers, are typically sized on the order of a few millimeters to
several tens of millimeters.
[0056] A titanium-sapphire (TiS) laser is a tunable laser capable
of emiting infra-red laser light with wavelengths ranging from
about 700 to about 1100 nm.
[0057] An yttrium-aluminum-garnet (YAG) laser, such as a neodimium
YAG, a horonium YAG, or an erbium YAG laser, is a solid-state laser
emitting laser light at a wavelength on the order of a micron.
Water molecules absorb energy at micron wavelengths; water
preferentially absorbs energy at wavelengths near 3 .mu.m, and
erbium-doped YAG lasers emit light with a wavelength of 2.94 .mu.m,
making them particularly suitable for use in photoablation by
rapid, local vaporization of water present in cells and tissues,
causing rapid expansion and ablation of tissue.
[0058] An ion beam is a beam of ionized gas molecules, typically
excited by radio-frequency energy and directed at a target. Ion
beam sources used in the practice of the present invention may be
of any kind; an ion beam source is described, for example, in U.S.
Pat. No. 5,216,330 to Ahonen. Ion beams may be used to create holes
in materials. U.S. Pat. No. 6,093,445 to Nawate describes an ion
beam method for producing rectangular and circular holes sized from
about 10 nm to about 2 .mu.m.
[0059] A tissue implant 10 having microfabricated lens capsule
tissue 12 with attached cells 14 and a dissolvable substrate 16 is
shown in cross-section in FIG. 1A. Cells 14 are attached to and
growing upon upper surface 18 of the microfabricated lens capsule
tissue 12. Lower surface 20 of the microfabricated lens capsule
tissue 12 is in contact with the dissolvable substrate 16. Cells 14
are iris pigment epithelial cells, which have apical 22 and basal
24 membranes, with basal membranes 22 in contact with upper surface
18 of the microfabricated lens capsule 12. Expression of the proper
cellular differentiation into basal 22 and apical 24 membranes, as
is found in pigment epithelial cells in vivo, is indicative of the
proper functioning of the epithelial cells growing on the
microfabricated lens capsule.
[0060] FIG. 1B illustrates, in cross-section, an eye 26 of a
mammalian animal into which the tissue implant 10 has been
surgically placed. FIG. 1C is a detail of the region within circle
33 of eye 26 including neural retina 28 and tissue implant 10.
Shown in FIGS. 1B and 1C are the neural retina 28, the iris pigment
epithelium (IPE) 30, the retinal pigment epithelium (RPE) 32
growing on Bruch's membrane 34 which separates the choroid 36 from
the basal membrane 38 of the RPE. The apical membrane 40 of the RPE
has numerous processes, which enfold and surround the
light-sensitive portions of the photoreceptors in the photoreceptor
layer 42 of the neural retina 28. The space between the apical
membranes of the RPE 40 and the photoreceptors 42 is the subretinal
space 44. The choroid 36 serves to maintain an environment capable
of supporting the high metabolic demands of the photoreceptor layer
42 in particular and the neural retina 28 in general by allowing
the passage of nutrients and electrolytes to, and removal of waste
products from, the subretinal space 44.
[0061] In a healthy eye, the subretinal space 44 is only a virtual
space, there being only minimal separation between photoreceptors
42 and apical portions of the RPE 40. However, in many eye
disorders, such as retinal detachment, the photoreceptors 42 may
become separated from the apical RPE membranes 40. In addition, the
neural retina 28 and the pigment epithelium 30 and 32 may be
artificially separated during eye surgery if desired. As shown in
FIGS. 1B and 1C, a tissue implant 10 may be placed into the
subretinal space 44.
[0062] Microfabricated lens capsule 12 with adherent IPE cells 14
is shown implanted into eye 26 where the IPE cells 30 are able to
contact photoreceptors 42 and provide metabolic support.
Dissolvable substrate 16 is shown between the lower surface 20 of
the microfabricated lens capsule 12 and the choroid 36, as it is
initially after placement of the tissue implant 10. However, the
dissolvable substrate 16 will dissolve and be removed from the
subretinal space 44 leaving only the transplanted microfabricated
lens capsule 12 and attached IPE cells 14 in place in the
subretinal space 44.
[0063] FIG. 2 is a scanning electron micrograph (SEM) of a
poly(dimethylsiloxane) (PDMS) microfabrication stamp embodying
features of the invention. The grid-lines are about 5 .mu.m wide
and are separated by about 50 .mu.m. The structural height of the
PDMS stamp is 7 .mu.m from the base to the face. Grid lines may be
coated with compounds, for example, poly-L-lysine, for placement
onto a surface by contacting the surface with the stamp. The
hexagonal shape was designed to mimic that of RPE cells in the
human macula.
[0064] FIG. 3 is a SEM of a microfabricated human lens capsule
tissue after contact with the microfabrication stamp shown in FIG.
2 that had been coated with a mixture of 2% polyvinyl aclohol (PVA)
and 0.1 mg/mL fluorescein. The bright areas show microprinting of
the fluorescein solution. Micropattern lines follow the same
pattern and spacing as the stamp that produced them by contact with
the lens capsule surface.
[0065] FIG. 4 shows a SEM of a PDMS stamp that has a stamp surface
with a topology given by an array of circular wells. Thus, the
stamp surface of stamp has circular depressions that will not
receive a coating, while the rest of the stamp surface does receive
a coating of molecules that may then be transferred to any surface
with which it becomes in contact. Coating the stamp with molecules,
such as PVA, mucilage, or other inhibitory molecules, and then
placing the stamp in contact with a surface, such as a lens capsule
surface, leaves a pattern of those molecules on the surface
everywhere but on the circles themselves. Such a micropattern of
inhibitory molecules allows cells growing on the surface to attach
only on these circular areas. Because the unmodified lens capsule
surface actively allows growth of cells adherent to it, inhibitory
patterns are required for patterned growth. Each circle is about 50
.mu.m in diameter.
[0066] FIG. 5 is a SEM of a human lens capsule that has received a
micropattern of PVA inhibitory molecules from the PDMS stamp as
illustrated in FIG. 3. The scale bar is 25 .mu.m. It is evident
that RPE cells are growing in a pattern determined by the
micropattern deposited on lens capsule by stamp. Cells remained
viable and in this pattern for as long as 24 days. It was found
that RPE cells on microprinted lens capsule after 24 days in
culture maintained the same pattern after this 24 days, were still
viable, and retained their cuboidal structure. By controlling the
width of the grid lines, cells can be separated to a greater or
lesser degree. Thinner grid lines allow growing cells to touch each
other, allowing formation of a transporting epithelial layer from
the contacting, growing cells.
[0067] Attachment of cells onto the microfabricated tissue
substrate may be speeded or enhanced by placement of the
microfabricated tissue within a flat-bottomed centrifuge tube along
with cells to be grown on the microfabricated tissue.
Centrifugation at low speed, such as, for example, between about
5,000 to about 15,000 revolution per minute rapidly deposit the
cells onto the microfabricated tissue and aid the directed growth
of deposited cells onto the microfabricated tissue.
[0068] Placement of microfabricated tissue onto, or coating a
microfabricated tissue with, a carrier matrix aids in its
processing and in its implantation into the body of an animal.
Microfabricated tissue may be coated on one side only, or, in some
embodiments of the invention, microfabricated tissue may be coated
on both sides. The processing and implantation of microfabricated
lens capsule, microfabricated inner limiting membrane,
microfabricated Bruch's membrane, or other microfabricated
membranous tissue may be aided in this way; for example, a carrier
matrix makes microfabricated tissues more rigid and easier to
handle. In addition, a carrier matrix is effective to prevent
folding and curling of the tissue, allowing implantation of a flat,
spread-out tissue sheet. Such a spread-out configuration provides
maximal surface area for growth of implanted cells, and provides
the implanted cells with maximal access to fluids and surrounding
tissues. Biodegradable carrier matrices embodying features of the
invention are flexible, fitting easily to the contours of the
retina. Preferably, the carrier matrix is biodegradeable, and so
may be resorbed by the host body within a desired time period after
placement in the eye. A desired time may be about a week to a few
months, preferably a few weeks to about two months, more preferably
a carrier matrix embodying features of the invention biodegrades
after implantation in a retina within about two weeks to about six
weeks.
[0069] Biodegradable matrix materials suitable for assisting in the
processing of tissues and in the implantation of tissues into the
eye include, for example: collagen; glycosaminoglycans; chitosan;
poly(hydroxyalkanoates); poly(oc-hydroxy acids), including but not
limited to polyglycolic acid (PGA), polylactic acid (PLA), and
polylactide-polyglycolide (PGA-PLA) mixtures, alloys and copolymers
(PLGA); poly(dioxanones); poly(.epsilon.-caprolactone); poly(ortho
esters); poly(anhydrides); poly(phosphazenes); poly(amino acids);
and other compounds, polymers, copolymers, alloys, mixtures and
combinations of these materials. FIG. 6 shows a lens capsule
(stained blue) on a carrier matrix of poly-lactide/polyglycolide.
The scale bar is 1 mm in length. Carrier matrix substrates and
coatings may be dyed (e.g., with trypan blue or rhadamine),
improving visualization of the tissue to be implanted during
implantation surgery. Such coatings and substrates may be used for
lens capsule, inner limiting membrane, Bruch's membrane, and other
membranous tissue, including corneal tissue, amniotic membrane,
serosal membranes, mucosal membranes, and neurological tissue.
[0070] FIG. 7 shows a section of rabbit retina containing a human
lens capsule on a poly-lactide/polyglycolide carrier matrix. The
retinal section shown was taken one week after implantation of the
lens capsule tissue in the subretinal space between the neural and
pigmented retinal cells in a rabbit eye. The lens capsule has a
flat configuration, showing no folding or curling that would
interfere with the flow of nutrients and waste products to and from
the transplanted cells.
[0071] Tissues to be modified may be obtained by means known in the
art, such as excision, biopsy, at surgery or at autopsy. As will be
understood by those of ordinary skill in the art, care should be
taken to avoid damage or contamination of the membranous tissue
during procedures for obtaining it, as by following standard
sterile operating procedures. It will be understood that the
methods and apparatus are suitable for modifying any membranous
tissue, including but not limited to ocular membranous tissue.
[0072] In the following, methods and apparatus for modifying tissue
are described using primarily lens capsule tissue as exemplary
membranous tissue. The methods and apparatus are thus also suitable
for modifying inner limiting membrane tissues and other tissues,
and may be used to modify inner limiting membrane and other tissues
as well. The tissue modification provided by the methods of the
invention is effective to alter the properties of the subject
tissue to provide a more favorable substrate for cell attachment
and growth, and to alter the physical and biochemical properties of
the lens capsule tissue to allow more ready exchange of fluid and
solutes across the tissue.
[0073] Membranous tissue such as lens capsule tissue and inner
limiting membrane may be obtained from donor eyes, or from the
patient (autologous tissue) by techniques known in the art, such as
following lens extraction for cataract surgery. For example, lens
capsule tissue may be obtained from an eye after a cataract
incision has been made (either a scleral incision or a corneal
incision). In this method, viscoelastic is next placed in the
anterior chamber following making an incision. The viscoelastic is
usually either Healon.RTM. (Pharmacia, Kalamazoo, Mich.) or
Viscoat.RTM. (Alcon, Fort Worth, Tex.). The capsulotomy is then
performed by using a cystotome needle. This needle is used to
puncture the anterior capsule centrally, creating a capsule flap.
This flap is then raised using the cystotome needle. Utrata forceps
are used to grasp the flap of the capsule and it is pulled in a
circular fashion. Pulling of the capsule for 360.degree. in a
controlled fashion will result in a round continuous capsulorhexis,
exposing the cataract. The lens and lens capsule may then be
removed.
[0074] Once removed, the membranous tissue (e.g., lens capsule,
inner limiting membrane, or other eye tissue) may be maintained in
vitro or prepared for in vivo transplantation. Membranous tissue is
then placed on a glass, plastic, or polymer substrate. The glass
substrate may be, for example, a glass cover slip. The plastic
substrate may be, for example, a tissue culture dish. The polymer
substrate, for example, may be a biodegradable polymer.
Biodegradable polymer films may include poly-lactic acid,
poly-glycolic acid, poly-lactic acid glycolic acid copolymers
(PLGA), including PLGA (50:50 lactic to glycolic acid copolymer),
poly-lactic acid polymers (PLLA), or polyethylene glycol/polylactic
acid copolymer (PEG/PLA), polyorthoesters, polyanhydrides,
polyphosphazines and blends and copolymers thereof. Methods for
using biodegradable polymer films may be found in, e.g., U.S. Pat.
No. 5,512,600 to Mikos et al.
[0075] For example, the methods discussed in U.S. Pat. No.
5,512,600 and in J. Biomedical Materials Research, Vol 34:87-93
(1997) by Giordano et al. may be used to maintain healthy lens
capsule, inner limiting membrane, or other membranous tissue in
vitro and in vivo. Biodegradable (e.g., dissolvable after placement
in an animal) polymer films comprising poly-lactic acid polymers
(PLLA), poly-glycolic acid polymers, polyorthoesters,
polyanhydrides, polyphosphazines, poly-lactic acid glycolic acid
copolymers (PLGA), including PLGA (50:50 lactic to glycolic acid
copolymer), and polyethylene glycol/polylactic acid copolymer
(PEG/PLA) films may be placed on the bottom of plastic petri
dishes. The lens capsule or other membranous tissue is then placed
onto the surface and smoothed down with the use of a pipette. The
membranous tissue and polymer film are transplanted together. The
film dissolves in vivo leaving the membranous tissue behind. The
film provides a greater ease of manipulation for the membranous
tissue; for example, polymer films prevent lens capsule from
curling, which is a problem observed with prior art methods. In
addition, further treatment of the membranous tissue may be applied
following these steps.
[0076] Lens capsule tissue (or other membranous tissue) may be
placed in an environment suitable for cell growth, such as a tissue
culture incubator or environmental chamber. In one embodiment, lens
capsule tissue is immersed in a phosphate buffered saline solution
(PBS) and maintained at 37.degree. C. in a 95% O.sub.2-5% CO.sub.2
atmosphere. Following incubation, the PBS is removed with a sterile
pipette and the lens capsule is allowed to lie flat on the bottom
of a sterile petri dish. The lens capsule is then soaked in
trypsin-EDTA for 1 hour to remove any lens epithelial cells and
subsequently, penicillin/streptomycin for 30 minutes for sterility.
The lens capsules are then rinsed three times in PBS followed by
three rinses in distilled water. Each rinse is performed carefully
with sterile pipettes. Finally, the lens capsule and the petri dish
it rests on are sterilized under UV light for at least three
hours.
[0077] In another embodiment, an interface chamber is used, wherein
lens capsule tissue (or other membranous tissue) is placed on
wetted filter paper covering a dish filled with phosphate buffered
saline, and maintained at 37.degree. C. in a 95% O.sub.2-5%
CO.sub.2 atmosphere. It will be understood that various saline
solutions known in the art, such as bicarbonate-buffered saline, or
other saline solutions, may be substituted for PBS. Alternatively,
culture medium (such as, for example, those as RPM1, DMEM or Hamm's
F12 (Life Technologies, MD)) may be added to or may replace the
saline in the methods, and growth factors, antibiotics, serum, and
other materials may be added to the saline or culture medium used
in maintaining lens capsule tissue.
[0078] Methods for modifying tissues include bulk modification
methods and surface modification methods. Bulk modification methods
include methods where substantial portions of the tissue, not
limited to surface portions of the tissue, are modified by the
method. Surface modification methods include methods wherein the
tissue is modified at and near to the surface of the tissue, but is
not greatly modified in other portions of the tissue.
[0079] The methods of the invention as applied to lens capsule
tissue, whether bulk or surface modification methods, include
removal of a lens capsule from an eye, flattening the lens capsule
onto on a sterile glass or plastic substrate, such as a culture
dish, microscope slide or a glass coverslip, that is submersed in
phosphate buffered saline or other suitable solution, followed by
further treatment of the lens capsule. It will be understood that
similar treatments may be applied to inner limiting membrane
tissue, Bruch's membrane, amniotic membrane, or other tissue.
[0080] Plastic substrates such as culture dishes and glass
substrates such as microsope slides may be sterilized by standard
procedures, such as by irradiation with ultraviolet light,
immersion in acid followed by repeated washing in sterile distilled
water, or other procedures known in the art. In addition, plastic
or glass substrates may be used with or without surface coatings.
Surface coatings may include collagen, collagen gel, fibronectin,
laminin, a silane coating such as polymethyl silane, a polymer
coating such as poly-L-lysine, or other coating known in the
art.
[0081] In embodiments of the invention, the substrate is prepared
for the membranous tissue. For example, tissue-culture plastic may
be rinsed in a 70% ethanol solution to remove dust and oils and
allowed to air dry. Following the drying step, the tissue culture
plastic may be covered with a solution comprising a desired
extracellular matrix molecule (e.g., 4 mg/ml collagen, type I rat
tail in PBS, 1 .mu.g/ml laminin from human placenta in PBS, or 25
.mu.g/ml fibronectin from human plasma in PBS) (collagen and
fibronectin may be purchased from Sigma, St. Louis, Mo.). After one
hour, the plastic may be rinsed in sterile distilled water twice
and allowed to dry under UV overnight. If the lens capsule
substrates are not immediately stamped, they are stored at
4.degree. C.
[0082] Bulk modification methods for modifying membranous tissue
such as lens capsule tissue include methods for modifying the
thickness, permeability, and other properties of the lens capsule
tissue. In one embodiment of the bulk modification method, such
further treatment includes use of an excimer laser to ablate the
surface of the lens capsule so that the overall thickness of the
lens capsule is reduced. For example, the lens capsule may be
ablated by a laser or ion beam, or by mechanical methods, so that
the overall thickness mimics the thickness of Bruch's membrane.
[0083] A laser, such as an excimer laser (e.g., an argon fluoride
laser (Lambda Physik, Model 201 E)) may be used to provide pulses
of laser light effective to ablate the surface of a lens capsule.
For example, pulse of between about 10 to 20 ns duration, delivered
at a frequency of about 1 to 50 Hz, with pulse energy densities of
between about 300 to 500 millijoules per square centimeter
(mJ/cm.sup.2) are effective to ablate the surface of a lens capsule
in a desired manner. Each pulse is effective to ablate the tissue
to a depth of between about 5 to 50 microns. Accordingly, repeated
pulses are effective to reduce the thickness of the lens capsule
tissue to a desired overall thickness. Methods as have been applied
to the cornea may be followed or adapted and are suitable for use
in photoablation of lens capsule tissue. Such methods of corneal
photoablation are disclosed in, e.g., U.S. Pat. No. 4,665,913 to
L'Esperance, U.S. Pat. No. 5,634,920 to Hohla, and U.S. Pat. No.
5,735,843 to Trokel.
[0084] In another embodiment of the bulk modification method, such
further treatment following placement of tissue on a glass
substrate includes use of a laser, such as, e.g., a YAG laser to
produce micropores in the lens capsule. Such bulk modification by
providing micropores alters the properties of the lens capsule
tissue so as to provide a more favorable substrate for cell
attachment and alters the biochemical properties of the lens
capsule tissue to allow more ready exchange of fluid and solutes
across the tissue. In embodiments of the invention, the micropores
are sized on the order of 10s of nanometers (nm) or less in
diameter. Thus, micropores produced by the bulk modification
methods may range in size between about 0.01 micron to about 10
microns, preferably between about 0.1 micron to about 1 microns. An
erbium YAG laser can be used to provide pulses of between about 10
to 50 ns duration, at energy levels of between about 1 to 50 mJ,
preferably between about 1 to about 20 mJ, effective to ablate
holes in lens capsule tissue according to the methods of the
invention.
[0085] In another embodiment of the bulk modification method, such
further treatment following placement of membranous tissue on a
glass substrate includes use of an ion beam to produce micropores
in the lens capsule to provide a more favorable substrate for cell
attachment and to allow more ready exchange of fluid and solutes
across the tissue. See, for example, Goplani et al. J Membr. Sci
118:93-98 (2000), Xu et al., in Material Research Society Symposium
Proceeding Vol. 540 "Microstructural Processes in Irradiated
Materials", pages 255-260 (1999), and Ohmichi et al., J. Nuclear
Materials 248:354-359 (1997). In embodiments of the invention, the
micropores are sized on the order of 10s of nms to a few Tm in
diameter.
[0086] The membranous tissue may be freeze dried for purposes of
exposing to the ion beams. Alternatively, the membranous tissue may
be dried out entirely, then rehydrated after the micropores are
made. An ion beam, such as a 120 MeV beam of Si.sup.28 ions, may be
used to irradiate the tissues. Following exposure to the ion beam,
the membranous tissues may be rehydrated. Biological etching using
collagenase and other proteases or proteolytic enzymes, as
discussed below, may be used to enlarge the microholes if larger
holes are desired.
[0087] In another embodiment of the bulk modification method,
treatment of the membranous tissue includes deposition of
proteolytic enzymes onto the membranous tissue effective to
biologically etch the surface and interior of the membranous tissue
to provide desired topology and surface adhesion properties to the
tissue. In some embodiments of this method, the deposition step
includes contacting the lens capsule or other membranous tissue
with a microcontact printing stamp carrying enzymes effective to
biologically etch the surface and interior of the tissue. After
stamping of the enzymes onto the tissue, albumin or an enzyme
inhibitor may be used to stop the reaction after a given time. For
example, incubation with collagenase is preferentially carried out
for various periods up to 26 h at 20.degree. C. in a constant
temperature water bath, and the collagenase reaction stopped by the
addition of EDTA to a final concentration of 50 mM. Incubation with
trypsin (e.g., 0.25% trypsin in a balanced salt solution without
calcium or magnesium) may be performed at about 0 to 5.degree. C.
for about 6 to about 18 hours. Following this incubation with
trypsin, the trypsin solution may be removed and the membranous
tissue incubated at 37.degree. C. for 20 to 30 minutes before
washing with a wash solution containing divalent cations (such as
calcium and magnesium) in the amount of about 1 to about 5 mM (and
optionally containing a trypsin inhibitor such as soybean trypsin
inhibitor). Alternatively, membranous tissues may be incubated with
dispase (about 0.5 to about 3 U/ml) or other proteolytic enzymes in
a balanced salt solution that is substantially divalent cation-free
at 37.degree. C. for up to several hours before removal of the
solution and washing of the membranous tissue with a balanced salt
solution containing about 1 to about 5 mM divalent cations.
[0088] In embodiments of the bulk modification methods, for
example, agents such as collagenase, trypsin, chymotryptsin,
dispase, liberase, thermolysin, pepsin, papain, and other proteases
may be applied as solutions in distilled water, phosphate-buffered
saline, or other buffered solution, at concentrations ranging
between about 0.01 mg/mL to about 100 mg/mL, preferably between
about 1 mg/mL to about 20 mg/mL, to the surface of a microcontact
printing stamp. The surface of the tissue, such as lens capsule
tissue, may be contacted in air or while immersed in a saline
solution. Where the protease is active in the absence of calcium,
such as for trypsin, chelating agents such as EDTA and EGTA,
preferably at concentrations in the range of between about 1 to
about 10 mM, may be included in the solutions. In such cases,
enzymatic action may be halted when desired by the addition of
calcium and or magnesium to the solution. In any case, enzymatic
action may be stopped by dilution with excess of enzyme-free
solution or by addition of an appropriate enzyme inhibitor. (For
example, trypsin may be inhibited by a trypsin inhibitor such as
soybean trypsin inhibitor (T-9003, Sigma Chemical Co. St. Louis,
Mo.)).
[0089] In another embodiment of the bulk modification method,
treatment of inner limiting membrane or lens capsule tissue
includes impregnation of the tissue with a deactivated enzyme, such
as a deactivated collagenase enzyme, that is activated by laser
light illumination. For example, in one embodiment very small
regions sized less than a micron in diameter of tissue are
activated by illumination with a 2-photon confocal laser system.
Enzymes activated in this way are effective to degrade or otherwise
alter tissue in the small region where activation occurs, while
nearby regions not activated by the confocal laser system remain
unaltered. The activated enzyme may be flushed out or deactivated
by water. Enzymes suitable for the practice of the invention
include but are not limited to collagenase, trypsin, chymotrypsin,
dispase, liberase, papain, pepsin, thermolysin, and other
proteases.
[0090] In one embodiment of the surface modification method,
microcontact printing techniques are used to fabricate chemical
micropatterns of biomolecules onto tissue. For example, surface
modification of lens capsule tissue may include deposition of
patterns of biomolecules onto lens capsule tissue. Such patterns
may include repeated iterations of geometric or linear patterns, or
may include only a few, or a single, pattern not made up of smaller
pattern units. Thus, patterns of surface modification may include
linear arrays of biomolecules deposited onto a tissue surface, or
curved arrangements of biomolecules, series of circularly-shaped
patterns, such as rings or dots, of biomolecules, or a series of
other shapes, including multiple shapes in a single pattern, of
biomolecules. Alternatively, such patterns may include extended
areas substantially covered by deposited biomolecules, or extended
areas substantially devoid of deposited biomolecules. It will be
understood that the methods include any suitable pattern comprising
lines, shapes, or regions of deposited molecules, including regions
devoid of deposited molecules situated between regions with
deposited biomolecules. Such micropatterns may, in general improve
cell attachment and growth on the modified membranous surface.
However, in embodiments of the invention, micropatterns are
produces where regions of the modified membranous tissue are
rendered less suitable, or unsuitable, for cell attachment and
growth. In this way, cell attachment and growth may be directed to
and limited to those regions of the membranous tissue that have not
been so treated.
[0091] Microcontact printing stamps may include the entire pattern
to be deposited onto target tissue, or may include a portion of the
desired pattern. Where the stamp includes a portion of the desired
pattern, multiple applications of the microcontact printing stamp
to the tissue surface are effective to provide a desired pattern of
biomolecules on the tissue surface. Where the stamp includes the
entire pattern, biomolecules may be deposited onto the microcontact
printing stamp itself in the desired pattern.
[0092] The patterns of biomolecules on a microcontact printing
stamp may be determined by directed placement of the biomolecules
on the stamp, or may be determined by the surface geometry of the
stamp. Where the pattern of biomolecules is determined by the
surface geometry of the stamp, the geometric pattern may include
locally-raised ridges, where contact of the stamp with a source of
biomolecules is effective to deposit such biomolecules onto the
raised surfaces, with substantially no biomolecules being deposited
on other, non-raised portions of the surface. In such a
microcontact stamp, the pattern of biomolecules deposited onto a
tissue would follow the pattern of the raised surfaces
Alternatively, the pattern may include depressions, valleys or
fissures, such as scratches made into a surface, where contact of
the stamp with a source of biomolecules is effective to deposit
such biomolecules onto a major portion of the surface, with
substantially no biomolecules being deposited on the depressed
portions of the surface. In such a microcontact stamp with
depressions, biomolecules would be deposited over a substantial
portion of the tissue, with regions substantially lacking deposited
biomolecules following the pattern of the depressed surfaces.
[0093] In some embodiments of this method, the patterns are sized
on the order of a few microns or less. Accordingly, in embodiments
of the surface modification methods of the invention, the
individual patterns of which the overall patterns are comprised may
range in size between about 0.1 micron to about 20 microns,
preferably between about 0.5 microns to about 5 microns.
[0094] Biomolecules suitable for deposition onto tissue surface
include proteins, peptides, organic molecules, oligosaccharides,
and small chain polymers, including but not limited to collagen,
hyaluronic acid, keratin sulfate, glycosaminoglycan,
methylacrylate, poly (methyl methacrylate), polystyrene,
poly(methyl styrene), polylysine, polylactic glycolic acid
(PLGA)-derivatized polylysine, polylysine peptides, and silane
polymers such as octadecyltrichlorosilane (OTS). Surface
modification comprising deposition of biomolecules is effective to
alter biological properties of the tissue, such as the ability or
ease of attachment by cells placed onto microfabricated tissues.
For example, deposition of hydrophobic molecules is effective to
deactivate selective cell attachment sites on lens capsule
tissue.
[0095] Microcontact printing stamps may be made of any material
capable of retaining a suitable pattern, such as glass, ceramic,
metal, plastic, polymer, or other material. In presently preferred
embodiments of the method, microcontact printing stamps include
poly(dimethylsiloxane) (PDMS), which is commercially available
(e.g., Sylgard 184 from Dow Corning, Midland Mich. 48640).
Microcontact printing stamps may be cast in PDMS from masters
containing desired patterns, such as, for example, a grid pattern
of lines. Alternatively, where the pattern to be formed is
determined by the pattern of deposition of biomolecules onto a
tissue, the stamp may include a simple surface, such as a flat
surface, suitable for carrying biomolecules. Such stamps may
include pins, slotted pins, bars or rods, for example, and may have
circular, triangular, square, rectangular, other polygonal or
irregularly shaped perimeters.
[0096] In embodiments of the surface modification method, the
surface of the lens capsule tissue is masked to cover part, but not
all, of the surface of the lens capsule tissue, and then irradiated
with ultraviolet (UV) radiation effective to denature the
extracellular matrix (ECM) of the exposed portions of tissue. This
deactivates molecules specific for cell adhesion, and to inhibits
or prevents cell adhesion and growth in the exposed, but not the
covered, regions. Thus, in this embodiment of the methods of the
invention, portions of the substrate are rendered unsuitable for
cell attachment and growth. In this way, growing cells can be
directed to desired regions, and away from undesired regions.
[0097] In embodiments of the invention, the entire substrate
surface may be deactivated to prevent attachment or growth of
cells, and then specific regions reactivated. By deactivating
proteins that are specific for cellular adhesion, the growth of
cells may be limited to confined regions. A deactivating substance
is one that prevents the attachment, the spread, or both, of
growing cells. For example, 0.2% polyvinyl alcohol (PVA) solution
and mucilage are effective deactivating substances.
[0098] A surface may be deactivated, and a portion of that surface
reactivated, by application of a deactivating substance to the
surface. For example, 0.2% PVA applied to the surface of the lens
capsule is effective to deactivate the surface of the lens capsule.
Exposure of the deactivated lens capsule surface to a micropattern
of light from an excimer laser is effective to ablate a
micropattern on the lens capsule surface. For example, a
micropattern may be produced on the lens capsule surface by
illumination of the lens capsule surface through an irradiation
mask. The ablated micropattern, by removing or altering the
deactivating substance, reactivates portions of the substrate to
allow cell growth and spreading into the ablated regions, thereby
directing cell growth to follow a desired pattern.
[0099] The masking step may include placement of a grid onto the
tissue, where the grid includes a material effective to prevent
irradiation of the surface by a source of radiation, such as UV
radiation. The grid may be made of materials including metal,
glass, plastic, ceramic, polymer, protein, or other material
effective to absorb or reflect UV radiation.
[0100] In an alternative embodiment of the masking method, the
masking step includes using microcontact printing techniques to
apply a pattern of protecting molecules onto the surface of the
lens capsule tissue effective to prevent ECM denaturation in
regions covered by the protecting molecules. Thus, the grid of a
masking step may include a coating on the surface effective to
screen the surface from irradiation. Such a coating may include a
protein, preferably one rich in tyrosine and other amino acid
residues that absorb ultraviolet light, a polymer effective to
absorb UV light, or a small molecule effective to screen UV light,
such as, for example, para-amino benzoic acid (PABA).
[0101] It will be understood by one of skill in the art that
surface modification methods and bulk modification methods may each
be applied to a single tissue. Thus, for example, the same lens
capsule tissue may be treated with both surface modification and
bulk modification methods effective to provide microfabricated lens
capsule tissue.
[0102] Microfabricated tissues are suitable substrates for growing
cells. A method for growing cells on microfabricated tissues
includes providing a microfabricated tissue produced by one of the
methods described above, and applying cells to the microfabricated
tissue. For example, the microfabricated tissue may include a
microfabricated lens capsule with a pattern on its surface, such as
a pattern of collagen, and the cells may include IPE cells, RPE
cells, stem cells, or other cells. In preferred embodiments of the
invention that includes autologous tissue and cells, the
microfabricated tissues and the cells are obtained from the same
animal.
[0103] The invention also provides methods for using
microfabricated tissues, including surgical methods for
transplanting microfabricated tissues into an animal. In preferred
embodiments, the methods for transplanting microfabricated tissues
into an animal include surgical methods for transplanting
microfabricated tissues into the eye of an animal. In most
preferred methods, the transplantation of microfabricated tissues
into the eye of an animal includes transplantation of
microfabricated lens capsule tissue near to or into the retina of
an animal. In some embodiments, the transplanted tissue further
includes cells grown on microfabricated lens capsule tissues. In
other embodiments, the transplanted tissue includes RPE cells, IPE
cells, stem cells, or other cells grown on microfabricated lens
capsule tissues. Alternatively, dissolvable polymer substrates may
be used for growing cells for transplantation. In further
embodiments, the transplanted tissue includes RPE cells, IPE cells,
stem cells, or other cells grown on microfabricated membranous
tissues or on dissolvable polymer substrates, where the cells and
tissues are taken from the same animal as the animal into which
they are transplanted (autologous tissue).
[0104] Methods for isolating or removing RPE cells from an eye may
be found in Pfeffer, B. A., Chapter 10, "Improved Methodology for
Cell Culture of Human and Monkey Retinal Pigment Epithelium,"
Progress in Retinal Research, Vol. 10 (1991) Ed. Osborn, N., and
Chader, J.; these methods may also be applied to IPE cells. The
cells may be removed from a donor eye, or from the intact eye of a
patient, including the eye that will ultimately receive a
transplant of microfabricated tissue with cells. Methods for
harvesting cells obtained in a biopsy, as for an autologous
transplantation procedure, may be found in Lane, C., et al. Eye
3:27-32 (1989). Further methods for procurement of RPE and IPE may
be found, e.g., in Abe et al., 1999, Thumann, et al., 1999; Lappas
et al., 2000; and in Thurmann et al., 2000.
[0105] The IPE cells, RPE cells, stem cells, or other cells may be
dispersed in saline, such as phosphate-buffered saline, at a
density of between about 10.sup.4 cells/mL to about 10.sup.7
cells/mL. Isolated RPE cells, IPE cells, stem cells or other cells
may be applied to microfabricated tissue, for example, to
microfabricated lens capsule tissue by gently pipetting a solution
containing IPE cells, RPE cells, stem cells or other cells onto the
microfabricated tissue immersed in PBS, followed by maintenance of
the cells and tissue at 37.degree. C. in a sterile 95% O.sub.2-5%
CO.sub.2 atmosphere for 12 hours. The PBS may be removed with a
sterile pipette and the lens capsule allowed to lie flat on the
bottom of a sterile petri dish or other container. The lens capsule
may then be soaked in trypsin-EDTA for 1 hour to remove any lens
epithelial cells and subsequently, penicillin/streptomycin for 30
minutes for sterility. Following this, the lens capsules may then
be rinsed three times in PBS followed by three rinses in distilled
water. Each rinse should be performed carefully with sterile
pipettes. Finally, the lens capsule and its support are sterilized
under UV light for at least three hours.
[0106] Before the application of cells, i.e. either in situ and/or
in vivo through transplantation, microfabricated tissues, such as
lens capsule, inner limiting membrane, Bruch's membrane, and other
tissues, may be modified and microfabricated as described above.
Alternatively, or in addition to such modification and
microfabrication, a microfluidic channel or pattern of microfluidic
channels may be placed onto a membrane surface to be modified, and
a suspension of cells or molecules may be delivered to the membrane
surface. For example, a microfluidic network as described by
Delamarche et al. (Science 276:779-781 (1997)), herein incorporated
by reference in its entirety, may be applied to a membrane surface
in order to modify the membrane. In such a procedure, a trough or
series of troughs may be formed in PDMS or other biocompatible
material, the troughs configured to form conduits upon placement of
the PDMS onto a membrane surface, with the membrane surface serving
as a conduit wall. Cells or biomolecules may be brought into
contact with the membrane surface by flowing a solution containing
the cells or biomolecules, or containing both cells and
biomolecules, through the conduits. The cells and biomolecules may
thus be deposited onto, or may otherwise modify, the exposed
surface of the membrane that forms a wall of the conduit.
[0107] Isolated RPE cells, IPE cells, stem cells, or other cells
may also be applied to a membranous tissue which has been partially
covered by a stencil. A stencil suitable for the practice of the
invention is configured with a pattern of holes or passages passing
through its surface. Such a stencil covers underlying membranous
tissue when the stencil is applied to a membranous tissue, while
the pattern of holes or passages is effective to leave portions of
underlying membranous tissue exposed. A stencil for
microfabricating tissue may have a rim thicker than the bulk of the
stencil in order to help provide mechanical strength. A stencil
having such a pattern of holes or passageways may be applied to a
surface of membranous tissue to be microfabricated, effective to
direct the growth of cells on the membranous tissue or to modulate
the exposure of the membranous tissue to external agents and
treatments. In some embodiments of this method, the patterns may be
sized on the order of a few microns or less, or on the order of
several microns, so that patterns may range in size between about
0.1 micron to about 100 microns, preferably between about 1 micron
to about 75 microns, more preferably between about 5 microns to
about 50 microns. A stencil may be formed of any suitable
biocompatible material, such as, for example, PDMS or PLGA/PEG
copolymer. A suitable stencil material may be solid or gelatinous,
and is preferably flexible. A stencil material may also be
biodegradable (e.g., PLGA/PEG copolymer). Stencils suitable for
application to membranous tissue for transplantation into the eye
of an animal may be made by methods similar to those described in,
for example, Folch et al. J. Biomed. Mater. Res. 52:346-353 (2000),
hereby incorporated by reference herein in its entirety.
[0108] Transplantation of microfabricated lens capsule tissue into
the subretinal space may be effected by any means providing access
to the subretinal space. Access to the subretinal space may be
provided, for example, by a scleral incision placed laterally on
the eye, or via the vitreous humor by a more frontal incision.
Procedures providing access to, and transplantation into, the
retina, including the subretinal space, have been described; see,
for example, Abe et al., Tohoku J. Exp. Med. 189:295-305 (1999),
Abe et al., Tohoku J. Exp. Med. 191:7-20 (2000), Lappas et al.,
Graefes's Arch Clin Exp Ophthalmol. 238:631-641 (2000), Thumann, et
al., Arch. Ophthalmol. 118:1350-1355 (2000), U.S. Pat. No.
5,962,027 to Hughes and U.S. Pat. No. 6,045,791 to Liu.
[0109] Alternatively, a microcontact printing stamp, or a stencil,
may be applied to an ocular membrane in vivo. For example, access
to Bruch's membrane within an intact, living eye may be
accomplished by standard surgical procedures, including formation
of a bleb by infusion of a gas, saline, mineral oil, or other
biocompatible liquid into the subretinal space of an eye, and
placement of a microcontact printing stamp or of a stencil onto
Bruch's membrane. Such application of a microcontact printing
stamp, or of a stencil, may be performed wet, that is in the
presence of normal bodily fluids, PBS or other artificial
physiological solution, mineral oil, or other biocompatible liquid.
Alternatively, such application of a microcontact printing stamp,
or of a stencil, may be performed dry, that is in the absence of
normal bodiy fluids or artificial physiological solution, by, for
example, filling the subretinal space with an inert gas such as
nitrogen or argon.
[0110] An intact Bruch's membrane may be prepared for in situ
microfabrication by scraping or otherwise debriding Bruch's
membrane to remove RPE cells before application of a stamp or a
stencil. A stamp or stencil may then be used to provide a desired
pattern onto a surface of the membrane. A PDMS stencil, for
example, having a pattern of passages may be applied to the surface
of Bruch's membrane; Cells are able to attach and grow on the
membrane surfaces exposed by the passages. Similarly, biomolecules
in a solution on contact with the membrane surface are able to
contact and modify or adhere to the membrane surfaces exposed by
the passages. The pattern of passages is effective to provide a
pattern suitable for directing the depostion of biomolecules, and
of directing the growth of added cells, such as RPE, IPE, stem
cells, or other added cells, or the regrowth of endogenous cells
from other regions of the eye. Alternatively, or in addition, a
microcontact printing stamp carrying laminin, fibronectin, or other
desired coating agent, may be applied to the ocular membrane
surface. In further alternative methods embodying features of the
invention, internal ocular membranes may be accessed via the
sclera, as, for example, by scleral puncture, scleral incision,
formation of a scleral window, or other method. In methods taking
advantage of scleral access, there may be no need to traverse the
vitreous humor in order to access ocular membranes for
treatment.
EXAMPLE 1
[0111] Microcontact printing was used to deposit micron-sized
patterns of biomolecules onto lens capsule tissue. Poly(dimethyl
siloxane) (PDMS) stamps were cast from masters containing a
topological pattern of grid lines spaced 50 microns apart. The PDMS
stamp was made from a master that was microfabricated from a
silicon wafer. PDMS stamps were used to microfabricate patterns
onto lens capsule tissue. Shown in FIG. 2 is a scanning electron
micrograph (SEM) of a PDMS stamp used to deposit a micropattern
onto a piece of human lens capsule tissue.
[0112] The PDMS stamp shown in FIG. 2 has a surface topology given
by a hexagonal array of 5 .mu.m-wide lines. Each line is separated
by approximately 50 .mu.m. FIG. 3 shows a human lens capsule
stamped with the PDMS stamp shown in FIG. 2. The PDMS stamp was
used to deposit hexagonal patterns of a PVA and fluorescein
solution (2% PVA and 0.1 mg/mL fluorescein) onto the lens capsule.
This example shows that the stamp is effective to produce a pattern
on a surface corresponding to the pattern of the stamp.
EXAMPLE 2
[0113] A SEM of a PDMS stamp with circular patterns used for
micropatterning tissue is shown in FIG. 4. As shown, the stamp has
a surface topology given by an array of circular wells of
approximately 50 .mu.m in diameter. When the relief pattern is
coated with an inhibitory molecule, such as PVA or mucilage, and
the stamp applied to a lens capsule, the inhibitory molecules are
transferred to the lens capsule in the pattern shown. FIG. 5 shows
the surface of a lens capsule that has been patterned with a PDMS
stamp having a pattern as shown in FIG. 2 and RPE cells grown on
it. This example shows that the stamp is effective to place a
pattern on the lens capsule surface that corresponds to the pattern
of the stamp, and for cell growth to be patterned according to the
pattern of the stamp.
[0114] Thus, application of the stamps of the invention are able to
deposit inhibitory molecules in patterns that can direct the growth
of cells growing on a patterned substrate. Because the lens capsule
actively allows growth, patterns of inhibitory molecules, such as
PVA, are preferred for patterned growth. Use of the stamp on
substrates treated to inhibit growth would require the use of
activating molecules to pattern growth on the substrate.
EXAMPLE 3
[0115] Masking of the surface of lens capsule tissue and then
irradiating the exposed surface, but not the masked surface, with
UV radiation is accomplished by placement of a SEM grid onto the
surface of lens capsule tissue. A SEM grid with spacing of 50
microns is placed onto the exposed surface of an excised lens
capsule tissue resting on a glass coverslip immersed in
phosphate-buffered saline. The surface of the lens capsule tissue
and the SEM grid are not immersed in the phosphate-buffered saline,
but rise above the level of the phosphate-buffered saline. UV light
is directed onto the exposed surface of the lens capsule tissue
effective to irradiate the lens capsule tissue not resting
immediately below the SEM grid material. After irradiation, the SEM
grid is removed. The lens capsule surface has a micropattern of
lines including tissue not irradiated (regions under SEM grid
material) enclosing regions comprising irradiated tissue.
EXAMPLE 4
[0116] Growth of monolayer cultures of retinal pigment epithelium
and iris pigment epithelium cells is facilitated by flat substrate
and by insuring that the substrate does not curl or fold upon
implantation in the subretinal space or other region of the eye. A
biodegradable matrix coating was found to prevent folding and
curling of lens capsule tissue. Such a biodegradable matrix
coating, which prevents substrate curling or folding is suitable
for use as a substrate for the growth of monolayer cultures of
retinal pigment epithelium and iris pigment epithelium cells for
implantation into an eye.
[0117] A biodegradable polymer matrix of poly(dl-lactide/glycolide)
was made by dissolving 50 mg of a 90:10 mixture of
poly(dl-lactide/glycolide) (Polysciences, Inc., Warrington Pa.
18976) in different amounts of dichloromethane to make solutions of
100 mg/mL (0.5 mL dichloromethane), 150 mg/mL (0.33 mL
dichloromethane), and 200 mg/mL (0.25 mL dichloromethane).
[0118] Human lens capsules obtained during cataract surgery were
stored in phosphate buffered saline at 4.degree. C. prior to
sterilization under ultraviolet light (254 nm for three hours) and
treatment with 0/05% trypsin-(ethylene diamine tetraacetic acid)
for ten minutes at 37.degree. C. to remove native epithelial cells.
Treated lens capsules were spread in a single layer on
Parafilm.RTM. (Pechiney Plastic Packaging, Inc., Neenah, Wis.
54956) in a Petri dish and coated on one side with the
poly-d-lactyl glycolic acid (PLGA) biodegradable matrix of a single
concentration by dispensing 5 .mu.L, 10 .mu.L or 20 .mu.L of the
PLGA solution from a pipette onto the lens capsule surface. The
PLGA solution was allowed to spread over a 5 mm diameter circular
area containing the flattened lens capsule. The solvent was
evaporated in a chemical hood.
[0119] Five New Zealand White rabbits weighing 2.5 to 3.5 kg
underwent implantation of the lens capsule/PLGA complex following
ketamine (40 mg/kg) and Xylazine (5 mg/kg) anesthesia. Tropicamide
0.5% and Phenylephrine 2.5% eyedrops were instilled into the
conjunctival sac of the left eye every five minutes for three
doses. Standard three-port pars plana vitrectomy was performed, and
a retinal bleb was inflated in the macular area by injection of
approximately 0.5 mL of balanced salt solution through a 42-gauge
needle. A retinotomy 1 mm in diameter was created, and the lens
capsule/PLGA was inserted into the subretinal space through the
retinotomy with subretinal forceps. The retina was then reattached
by air-fluid exchange.
[0120] The operated eyes were removed one week after implantation
of the lens capsule/PLGA and fixed in 1.25% glutaraldehyde/1%
paraformaldehyde in cacodylate bufer (pH 7.4). The eyes were then
cut open, fixed, post-fixed in osmium tetroxide, dehydrated in a
graded series of ethanol, embedded in epoxy resin, cut into 1 .mu.m
sections and stained with toluidine blue.
[0121] FIG. 6 shows a photomicrograph of a microfabricated lens
capsule on a polylactide/polyglycolide carrier matrix. Histological
studies performed one week post-implantation demonstrated that the
lens capsule remained flat on Bruch's membrane. This is illustrated
by a section of a rabbit retina having a lens capsule/PLGA implant
taken 1 week after implantation is shown in FIG. 7. There was local
disruption of the photoreceptor layer, and an overlaying rtinal
detachement, presumably in the area previously occupied by the
bleb. The PLGA was almost completely dissolved in all cases. The
five implantations demonstrated that lens capsule coated with PLGA
was easy to handle during surgery, and had sufficient rigidity so
that the implants remained flat within the subretinal space. No
evidence of significant inflammatory reaction was noted.
[0122] This example shows that PLGA greatly improves the surgical
handling of lens capsule during subretinal implantation and allows
the lens capsule to be implanted without curling. While untreated
lens capsule may roll into multiple layers or fold during
implantation, lens capsule treated with a bioabsorbable matrix
coating such as the PLGA coating used in this example remains
relatively flat in the subretinal space after implantation. Because
the PLGA degrades within a few weeks, concerns for late immune
reactions to an implant are allayed. This example demonstrating
improved mechanical characteristics of coated microfabricated lens
capsule illustrates that coating microfabricated tissues such as
lens capsule, inner limiting membrane, Bruch's membrane, and other
tissues, overcomes the limitations of the mechanical weakness of
the untreated tissue and provides an improved substrate for
implantation of tissues and cells.
EXAMPLE 5
[0123] One therapy for AMD is to transplant suspensions of either
RPE cells or IPE cells to rescue the diseased retina. The present
invention provides novel tissue engineering techniques to precision
engineer autologous human tissues as a substrate for transplanting
cells, such as IPE cells, RPE cells, stem cells, and other cells.
Suitable tissues include membranous tissues, such as lens capsule
(e.g., human lens capsule), inner limiting membrane tissue, Bruch's
membrane tissue, corneal tissue, amniotic membrane tissue, serosal
membrane tissue, mucosal membrane tissue, and neurological
tissue.
[0124] A microgeometry of inhibitory molecules is arranged onto the
surface of a suitable substrate. Suitable substrates include human
lens capsule, collagen gel, collagen-, fibronectin-, and
laminin-coated plastic, and a dissolvable polymer such as PLGA or
PLLA. Human lens capsules may be obtained during cataract surgery.
Cultures of experimental RPE cells are grown on these
microengineered surfaces and analyzed using scanning electron
microscopy, atomic force microscopy, and fluorescence microscopy.
Comparisons between microfabricated surfaces of autologous tissue
and synthetic surfaces and membranes are then made.
[0125] These comparisons demonstrate that individual RPE cells may
be directed to grow in microenvironments on the respective
biological surfaces. Although all surfaces studied are amenable to
micromachining, including human lens capsule, it will be understood
that different tissue engineering methods may be used to vary
cell-to-cell distance and the microenvironment of growth factors
and cell adhesion molecules.
EXAMPLE 6
[0126] In this example, isolated RPE cells are applied to a lens
capsule membrane tissue which has been partially covered by a
stencil and their growth on the lens capsule is directed by the
stencil pattern. Fibronectin (from human plasma, 25 .mu.g/ml in
PBS) is coated onto an excised lens capsule tissue resting on a
glass coverslip immersed in phosphate-buffered saline. A PDMS
stencil having a regular pattern of hexagonal holes of about 50
.mu.m across, the holes being spaced about 10 .mu.m apart, is
sterilely placed onto the excised lens capsule tissue. The
fibronectin promotes the adherence of the stencil to the lens
capsule as well as promoting the adherence of added cells. RPE
cells dispersed in cell growth medium (10.sup.7 cells/mL) are
sterilely added to the saline solution onto the surface of the lens
capsule that is partially covered by the stencil. The lens capsule,
stencil, glass coverslip, added cells, and cell growth medium are
maintained in a tissue culture dish in a tissue culture incubator
and are maintained under suitable culture conditions at
approximately 37.degree. C. in a 95% O.sub.2-5% CO.sub.2
atmosphere. The stencil's pattern of hexagonal holes leaves
portions of underlying membranous tissue exposed to the RPE cells.
The RPE cells adhere to the exposed lens capsule tissue, and grow
on it in hexagonal patterns directed by the stencil. The resulting
microfabricated lens capsule tissue with adherent RPE cells is
suitable for transplantation into the eye of an animal.
EXAMPLE 7
[0127] In this example, a microcontact printing stamp is applied to
an ocular membrane in vivo, providing a microfabricated membranous
surface suitable for growth of cells. Such a microfabricated
membranous surface suitable for the growth of cells aids in the
treatment of eye diseases or conditions stemming from defects of
ocular membranes or ocular cells. For example, in an eye having a
region of diseased RPE cells, or RPE cells which are not
functioning properly, removal of the defective RPE cells and
microfabrication of the underlying Bruch's membrane, optionally
with the addition of cells, is effective to treat the eye.
[0128] A subretinal bleb is used to access a retinal region in an
eye of a patient having diseased RPE cells. The bleb is formed by
infusion of sterile saline into the subretinal space following a
3-port pars plana vitrectomy and puncture of a small pathway
through the neural retina. RPE cells exposed by the bleb are
removed from a portion of Bruch's membrane by scraping or otherwise
debriding Bruch's membrane, such as by techniques used in choroidal
neovascular surgery. A microcontact printing stamp having a pattern
of 50 .mu.m-diameter circles spaced 10 .mu.m apart is coated with
25 .mu.g/ml fibronectin from human plasma in PBS, rolled into a
tubular configuration, and inserted into a needle. The needle is
connected to a syringe containing PBS. The microcontact printing
stamp and PBS are gently injected into the bleb within the
subretinal space, and the stamp unrolled by action of the needle
and of PBS delivered by the needle. The microcontact printing stamp
is placed in contact with the exposed Bruch's membrane. Such
placement of a coated microcontact printing stamp is effective to
deposit a pattern of fibronectin onto the exposed Bruch's membrane
and to prepare the intact Bruch's membrane in situ for
microfabrication effective to provide a pattern suitable for
directing the growth of added cells or the regrowth of endogenous
cells from other regions of the eye. After 15 minutes, the
microcontact printing stamp is removed from the bleb via the
pathway through the neural retina. In alternative treatments, the
microcontact printing stamp is left in contact with Bruch's
membrane for periods varying between about 1 minute up to about 1
hour. Following removal of the microcontact printing stamp, a
dispersion of RPE cells in PBS (10.sup.6 cells/mL) is gently
infused into the bleb. In further alternative treatments, in which
the microcontact printing stamp is made of biodegradable materials,
the microcontact printing stamp is not removed, but may remain in
place as cells are added and afterwards. The instruments are then
removed from the eye, the incisions are closed and standard
post-operative care is given to the patient. The RPE cells
proliferate to cover the exposed portion of Bruch's membrane and
aid in maintaining the health of Bruch's membrane and in the
support of overlying neural retina.
EXAMPLE 8
[0129] In this example, a stencil is placed onto an ocular membrane
in vivo, providing a microfabricated membranous surface suitable
for growth of cells. A subretinal bleb is used to access a retinal
region in an eye of a patient having diseased RPE cells. The bleb
is formed by infusion of sterile saline into the subretinal space
following a 3-port pars plana vitrectomy and puncture of a small
pathway through the neural retina. RPE cells exposed by the bleb
are removed from a portion of Bruch's membrane by scraping or
otherwise debriding Bruch's membrane, such as by techniques used in
choroidal neovascular surgery. Fibronectin (from human plasma, 25
.mu.g/ml in PBS) is diffused into the bleb to coat the exposed
Bruch's membrane and to promote adherence of an added stencil. A
stencil made from PDMS and having a pattern of hexagons measuring
60 .mu.m across at their widest dimension, and spaced 10 .mu.m
apart is rolled into a tubular configuration in PBS and inserted
into a needle. The stencil and PBS are gently injected into the
bleb within the subretinal space, and the stencil unrolled by
action of the needle and PBS delivered by the needle. The stencil
is placed onto the exposed Bruch's membrane. Such placement of a
stencil onto Bruch's membrane in situ is effective to provide a
pattern on the exposed Bruch's membrane effective to direct the
growth of added cells or the regrowth of endogenous cells from
other regions of the eye. Following placement of the stencil, a
dispersion of IPE cells in PBS (10.sup.5 cells/mL) is gently
infused into the bleb. The instruments are then removed from the
eye, the incisions are closed and standard post-operative care is
given to the patient. The IPE cells proliferate to cover the
portions of Bruch's membrane exposed by the gaps of the stencil and
aid in maintaining the health of Bruch's membrane and in the
support of overlying neural retina.
* * * * *