Note: Descriptions are shown in the official language in which they were submitted.
CA 02161659 2003-08-13
?~,TINAL PXGMENT EFITFiELit~'~~ TRANSPLA,NTATIOti
BACKG~(.Tt4D Off' 'iHE INVEN'jT,',IyO_N
'zhe pteSS~nt invention relates in general to cell
and t;ssue transplantation techniques. Mar$ particular:;,
the present invention is directed to techniques for
transplanting populations of retinal pigment epithelium
tRPE) cells as a monolayer to the subre:inal region ef the
eye, and to metha3s ~or pzeparing implants comprising
monolayers of RFE cells for transplantatiLn.
l~ Tha retina is the sensory epithelial surface that
lines the posterior a9peCt of the sYe. recelvss the image
formed by the laps, traasducea this image into neural
impulses and conveys this infonaation to the brain by the
optic nerve. The retina comprises a number of Isyers,
2o namely, the ganglion cell layer, inner plezitorm layer.
inner nuclear layer, outer plexiform layer.. outer nuclear
layer. photoreceptcr inner segments and outer segments.
The outer nuclear layer Coeapriats the cell bodies of thQ
photoreceptor cells with the inner and outer segments being
2~ eztensions of the Cell bodies. ThQ choraid is a vascular
raemhran~ containing large bfanchod pigment cells that lies
between the retina and the sclerotic Coat of the vertebrate
eye. atop thQ choraid is a membrane 1-3 microns in
thickness essentially composed of collag~n. known as
30 8ruch' s mecabrane.
- ~Im~nediately between Bruch's membrane and the
retina is the retinal piQraent epithelium which ~orms an
intimate stzLCtural arid functional relationship ~rith the
WO 94/25569 ~ PCT/US93/04245
2
photoreceptor cells. Among the functions performed by RPE
cells is the phagocytosis of outer segment debris produced ,
by the photoreceptors. It is believed that failure of the
RPE cells to properly perform their functions such as ,
digestion of outer segment debris leads to the eventual
degeneration and loss of photoreceptor cells.
In the leading causes of visual impairment in
western industrialized countries, such as age-related
macular degeneration (AMD), both photoreceptors and the
underlying RPE are compromised, or have degenerated. A
further aspect of ANm is the frequent appearance of
subretinal neovascular membranes which grow through the
Bruch's membrane and the RPE and tend to hemorrhage and
leak fluids into the subretinal space.
In an effort to recover what was previously
thought to be an irreparably injured retina, researchers
have suggested various forms of grafts and transplantation
techniques, none of which constitute an effective manner
for reconstructing a dystrophic retina. The
transplantation of retinal cells to the eye can be traced
to a report by Royo et al. , r w h ~: 313-336 (1959) in
which embryonic retina was transplanted to the anterior
chamber of the maternal eye. A variety of cells were
reported to survive, including photoreceptors.
Subsequently del Cerro was able to repeat and extend these
experiments (del Cerro et al., Invest. Ophthalmol. Vi,s.
1182-1185, 1985). Soon afterward Turner, et al.
Dev. Brain Res. x:91-104 (1986) showed that neonatal
retinal tissue could be transplanted into retinal wounds.
Li and Turner, Exp. Eye Res. x:911 (1988) have
proposed the transplantation of retinal pigment epithelium '
(RPE) into the subretinal space as a therapeutic approach
in the RCS dystrophic rat to replace defective mutant RPE '
cells with their healthy wild-type counterparts. According
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3
to their approach, RPE were isolated from 6- to 8-day old
black eyed rats and grafted into the subretinal space by
using a lesion paradigm which penetrates through the sclera
and choroid. A 1 ml bolus injection of RPE (40,000 -
60,000 cells) was made at the incision site into the
subretinal space by means of a 10 ml syringe to which was
attached a 30 gauge needle. However, while this technique
is marginally appropriate for immature RPE cells, with
mature cells it leads to activation and transformation of
these cells which damages eye and retinal tissue.
Lopez et al., Invest. Ophthalmol. Vis. Sci. ~Q:
586-589, 1989, also reported a procedure for the
transplantation of dissociated RPE cells. In this
procedure, RPE cells were obtained from normal, congenic,
pigmented rat eyes by trypsin digestion. These freshly
harvested, dissociated RPE cells were injected into the
subretinal area of the eyes of dystrophic RCS rats via an
incision through the sclera, choroid and neural retina.
Comparable to the Li and Turner approach discussed above,
this procedure destroys the organized native structure of
the transplanted RPE cells, which take the form of a
confluent monolayer in a healthy eye. Moreover, the
procedure is of questionable value for the transplantation
of mature RPE cells. When mature RPE cells are
transplanted in dissociated form, experimental results
indicate that they are likely to become activated, migrate
into the subretinal space, and as noted by Lane, C., et
al . , ~ ( 1989) ,~,, 27-32, invade the retina and vitreous .
This activation of the transplanted, mature RPE cells can
result in such pathologies as retinal pucker, massive
subretinal fibrosis, retinal rosette formation, retinal
detachment, and proliferative vitreoretinopathy.
" The difficulties discussed above associated with
the transplantation of mature RPE cells is problematic for
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4
human transplantation since available supplies of immature
human RPE donor tissue are extremely limited. Moreover, ,
the inability to use mature cells effectively prevents
transplantation using autologous RPE tissue, which ,
otherwise would be desirable to avoid the complications
involving potential immunological responses faced by
non-autologous transplants. Since the victims of AMD are
predominantly older adults, in most cases utilizing
autologous tissues for transplants would necessarily entail
the use of mature human RPE cells.
It is believed by the present inventor that it is
necessary to maintain adult human RPE cells substantially
as a monolayer to achieve their successful transplantation
and to avoid the problems associated with activation of RPE
cells. Although not wishing to be limited to a particular
theory, it is thought that the cell-to-cell contact
inhibition provided by an intact monolayer, supplemented by
adherence to a substrate, mitigates against RPE cell
activation. Moreover, a monolayer structure for the RPE
provides a proper foundation for the maintenance of the
photoreceptor cells in an organized outer nuclear layer
structure and for proper growth and arrangement of inner
and outer segments, believed by this inventor to be
advantagous to restore a reasonable degree of vision. The
requirement that the photoreceptors be maintained in an
organized structure is based on the well known optical
characteristics of photoreceptors (outer segments act as
light guides) and clinical evidence showing that folds or
similar, even minor, disruptions in the retinal geometry
can severely degrade visual acuity.
Additionally, in cases of AMD where subretinal
neovascular membranes have appeared, prior to RPE
transplantation, such membranes will need to be removed to
prevent subret~.nal edemas and hemorrhaging of these
94/25569 PCT/US93/04245
membranes. In practice, removal of the neovascular
membrane results in removal of the native RPE and Bruch's
membrane as well.
A critical impediment to the transplantation of
5 RPE cells as a monolayer is the fragility of the
intercellular structure of RPE relative to the rigors of
manipulation during transplantation to the subretinal
area. Moreover, providing satisfactory support to the RPE
cells during this process is complicated by the fact that
the support must either be removed subsequent to
transplantation, to avoid compromising metabolic exchange
between the choroid and the overlying retina, or be
compatible with such ongoing physiological activity. Thus,
a method is needed wherein an implant comprising a
monolayer of RPE cells is prepared and transplanted in
which the component supporting the monolayer of RPE cells,
upon transplantation to the subretinal area and exposure to
a set of predetermined conditions, does not impede normal
eye tissue function. Further, a Bruch's-like membrane for
attachment of the transplanted RPE cells will be needed in
those cases where neovascularization has occurred and the
native Bruch's membrane is removed.
SUN~IARY OF THE INVENTION
Among the objects of the present invention,
therefore, may be noted the provision of a method for
preparation of a population of RPE cells as a monolayer for
use in the reconstruction of a dystrophic RPE; the
provision of such a method which allows for use of mature,
and in particular, autologous, mature RPE cells as donor
tissue in the reconstruction of a dystrophic RPE; the
provision of an implant for use in the reconstruction of a
dystrophic retina; the provision of such an implant which
does not interfere with normal eye tissue function after
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6
transplantation and maintains photoreceptors and their inner and outer
segments by
allowing for maintenance of the native organization of the photoreceptor; the
provision of
such an implant which provides a membrane for attachment of the population of
RPE cells
especially when the native Bruch's membrane has been removed; and the
provision of a
method for transplantation of such implants to the subretinal area of an eye.
Briefly, therefore, the present invention is directed to a method for the
preparation
of a retinal pigment epithelial cell graft for transplantation to the
subretinal space of a
host's eye comprising the steps of:
a) providing donor tissue comprising retinal pigment epithelial cells;
b) harvesting retinal pigment cells epithelial from the donor tissue;
c) culturing the harvested retinal pigment epithelial cells on a culture
substrate to
form a monolayer said substrate being transplantable to the subretinal space
and upon
transplantation will not impede normal eye tissue function; and
d) apposing the cultured retinal pigment epithelial cell monolayer to a non-
toxic
flexible support that, upon transplantation to the subretinal space, will not
impede the
host's normal eye tissue function nor will it impede the normal eye tissue
function of the
transplanted retinal epithelial cells.
The invention further provides a method for the preparation of a population of
Retinal Pigment Epithelium cells for transplantation to the subretinal area of
an eye of a
host comprising:
a) providing donor tissue comprising Retinal Pigment Epithelium cells;
b) harvesting Retinal Pigment Epithelium cells from the donor tissue;
c) culturing the harvested Retinal Pigment Epithelium cells upon a culture
surface
having thereon a substrate suitable for attachment and growth of a monolayer
of Retinal
Pigment Epithelium cells, wherein said substrate can be removed along with
said
monolayer;
d) removing the monolayer of cultured Retinal Pigment Epithelium cells with
the
substrate from the culture surface;
e) apposing the monolayer of cultured Retinal Pigment Epithelium cells to a
non-
toxic flexible support that, upon transplantation to the subretinal area and
exposure to
conditions which result in a breakdown of at least a portion of the non-toxic,
flexible
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6a
support, will not impede normal eye tissue function of the eye of the host and
the
transplanted population.
The present invention is further directed to a graft or an implant for
transplantation
to the subretinal area of a host eye. The implant comprises a laminate of a
monolayer of
retinal pigment epithelium cells and a non-toxic, flexible support that, upon
transplantation
to the subretinal area and exposure to a set of predetermined conditions, will
not impede
normal eye tissue function.
The present invention is also directed to a method for transplanting to the
subretinal area of a host's eye an implant comprising a monolayer of RPE
cells. The
method comprises providing an implant comprising a laminate or a monolayer of
retinal
pigment epithelium cells and a non-toxic, flexible support that, upon
transplantation to the
subretinal area and exposure to a set of predetermined conditions, will not
impede normal
eye tissue function, making an incision through the host's eye, at least
94!25569 PCT/US93/04245
7
partially detaching the retina to permit access to the
subretinal area, and positioning the implant in the
accessed subretinal area.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a photograph (200X) of a cryostat
section of a normal rat retina and sub-retinal area;
Fig. 2(a, b, c, d and e) is a schematic showing
the preparation and transplantation of a preferred
embodiment of an implant comprising a monolayer of RPE
cells mounted to a support;
Fig. 3 is a schematic of an alternative
embodiment of an implant comprising a monolayer of RPE
cells mounted to a support;
Fig. 4 is a horizontal section through an eye
illustrating a transchoroidal and sclera! surgical approach;
Fig. 5 is a horizontal section through an eye
illustrating a transcorneal surgical approach;
Fig. 6 is a photograph showing a view of the
transplanted implant of an intact monolayer of mature human
RPE cells positioned under the retina of a rodent whose
cornea, pupil and lens have been removed, 2 weeks
post-transplantation, as set forth in Ezample 1;
Fig. 7 is a photograph (40X) of a section of a
rat retina and sub-retinal area showing an implant of an
intact monolayer of a population of mature human RPE cells
14 days after transplantation, as set forth in Ezample 1;
Fig. 8 is a higher magnification photograph
(400X) of a section of a rat retina and sub-retinal area
showing an implant of an intact monolayer of a population
of mature human RPE cells 14 days after transplantation, as
set forth in Ezample 1;
Fig . 9 is a photograph ( 100X) of a section of a
rat retina and sub-retinal area 14 days after
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transplantation of dissociated mature human RPE cells, as
set forth in Example 2; and
Fig. 10 is a higher magnification photograph
(300X) of a section of a rat retina and sub-retinal area 14 ,
days after transplantation of dissociated mature human RPE
cells, as set forth in Example 2.
DETAILED DESCRIPTION
As used herein, the term "donor" shall mean the
same or different organism relative to the host and the
term "donor tissue" shall mean tissue harvested from or
cultured from tissue harvested from the same or different
organism relative to the host. Autologous tissue shall
mean tissue harvested from or cultured from tissue
harvested from the host organism. Mature RPE cells shall
mean differentiated RPE cells derived from an organism that
is not a fetus.
It is believed that in age-related macular
degeneration (AMD), compromise or degeneration of the RPE
cells which underlie and form a close structural and
metabolic support for the photoreceptors, leads to the loss
or destruction of viable photoreceptors. It has been
discovered, however, that transplantation of a population
of RPE cells as a monolayer, i.e., in essentially a
two-dimensional array of cell bodies capable of engaging in
cell-to-cell contact inhibition, allows the RPE cells to
maintain largely normal characteristics of native, healthy
retinal pigment epithelia, capable of providing structural
and functional support to photoreceptor cells. Moreover,
as illustrated in Example 2 below, it is believed that it
is essential to transplant mature RPE cells as a monolayer
in order to prevent activation of such cells. Activation
leads RPE cells to transdifferentiate into wandering '
macrophages, fibroblast-like cells and other cell types,
94/25569 PCT/US93/04245
9
which cause a number of dystrophic effects in the eye and
retina.
Fig. 1 is a photograph of a cryostat section of a
normal rat retina and subretinal area. In Fig. 1 as well
as subsequent figures, the retina or layers thereof, e.g.,
the ganglion cell layer ("G"), inner plexiform layer
("IPL"), inner nuclear layer ("INL"), outer plexiform layer
("OPL"), outer nuclear layer ("ONL"), inner segments
("IS"), outer segments ("OS"), and retinal pigment
epithelium ("RPE"), are shown, respectively, from top to
bottom.
Referring now to Figs. 2 and 3, implants
comprising a monolayer of RPE cells are prepared, in
general, by harvesting RPE cells from donor tissue and
apposing the harvested RPE cells as an intact monolayer to
a non-toxic, flexible composition, or by seeding such a
composition with a monolayer of dissociated RPE cells and
allowing them to grow into a confluent layer. The flexible
composition serves as a stabilizing support for the RPE
cells during transplantation.
The implant constitutes a laminate of a monolayer
of RPE cells and a support composition approximately 100 to
500 microns thick, and preferably 150-250 microns thick.
The surface of the implant has a surface area greater than
about 1 square millimeter, preferably greater than 2 square
millimeters, and most preferably greater than 4 square
millimeters, or as large as may be, practically handled.
Thus constructed, the implant may subtend a considerable
extent of the sub-retinal surface.
In selecting donor tissue for the harvesting of
RPE cells, it is noted that previously, transplantation of
RPE cells was effectively limited to immature cells.
Mature RPE cells transplanted according to prior art
methods have been shown to undergo activation resulting in
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to
retinal pucker, sub-retinal fibrosis and p:olif3rati~e
vitreoretiropathy. However, by utilizing the proc~dures
for implanting a monolayer of FtPE cells according to this
invention, transplanted mature r~PE cells have successfully
maintained essentially normal structure and furi~tion.
Thus, the subject invention makes mature RpE cells
available for transplantation, significantly increasing tha
supply of usaDla donor tissue beyond the narzowl;r limited
supply of iammature human donor tissue.
l0 It is also noted that the RPE forms part o~ the
blood-retinal barrier and is thus a=posed to lymphocytic
attack. accordingly, use of a~utologous iiPE cells, now
possiblQ even far nature APE cells, is preferable, siaca it
will avoid immunolcgical complications is clinical
applications. If aan-autciogous tlsaLg is util=aed, tissue
typing and/or use of an immuaosuppreasion regimen will
generally be neceaaary to avoid rejaction upon
transplantation,
Donor tissue may ba provided. foe r_on-autologous,
human RP. tissue, from eye banks. which make the RPE tissue
available in conneetiari with ronductinq corneal
transplants. Harvesting donated tissue comprising
non-autalogous RPE cells can be accomplished by any
suitable method. In general. harvasting of RF~L cells may
z5 be carriad out as set torts 3n Pfeffer, s.A. , Chapter 1~.
"Improved i~8thodology for Cell Culture of ~tuman and rlonxey
Retinal Pigment Epitheliuat", proqrss_~ in R~ti~zal Research,
Vol. 10 ;1.991); Ed. by Osborn, H. and Chader, .:., or
Mayerson. P.L., at al., °'Aa Imprcvad Method for Isolation
sad Culture of Rlt Retinal Pigment Epitkselial Coils",
T"pv~st~qative O~,~ala~nl~,Qy & zjysuaS 9Cience, POV. , _ 1985;
~: 1599-I6fl9,
Specifically, to harvest non-autologeus RBE cells, a donor
eye is Finned by the optic nerve stump into an eye cage aa~
94!25569 PCT/US93/04245
11
placed in an eye jar immediately after corneal removal.
The jar is then flooded to capacity with cold Delbecco's
modified essential medium (DMEM) supplemented with 5% fetal
bovine serum. After loose connective tissue and muscle
have been carefully trimmed from the eye, it is placed
upright on a sterile plate and the anterior segment with
the adherent vitreous is lifted out of the eye cup. The
neural retina is separated at the optic disc and removed.
The shell is then washed with Hanks° balanced salt
solution, Ca++ and Mg++ free. and treated with 0.25%
trypsin for 30 min. at 37° C. The trypsin solution is
aspirated from the shell and DMEM (GIBCO) is added. The
RPE cells are released from Bruch's membrane by gently
pipetting the culture medium in the shell.
For implants containing autologous tissue, RPE
cells may be harvested by performing a biopsy following the
procedures disclosed by Lane, C., et al. in ~yg (1989) 3,
27-32.
The implant also comprises a support for the RPE
cells so that the monolayer of RPE cells is less likely to
be damaged and is more easily manipulated during the
transplantation process. The support consists of a
substrate, an overlayer or both, comprising a sheet or
sheets of a non-toxic, flexible composition selected to
provide mechanical strength and stability to the easily
damaged monolayer of RPE cells. Because the support
composition will be inserted into the eye as a laminate
with the monolayer of RPE cells, the support is also
comprised so that, upon transplantation to the subretinal
area and exposure to a set of predetermined conditions,
described herein, the support composition will not impede
normal eye tissue function of the host eye or the
population of transplanted RPE cells. If the harvested RPE
cells are to be cultured before transplantation, the
WO 94/25569 PCTlUS93/04245
12
composition providing support to the monolayer of RPE cells
during transplantation may optionally be capable of serving
as the attachment substrate for the RPE cells during
culturing.
A variety of compositions may be used as the
support for the population of RPE cells, depending upon the
specific set of conditions to which the composition will be
exposed after transplantation. In short, the composition
is selected either because any portion remaining within the
subretinal area for more than approximately one week after
transplantation is compatible with normal eye tissue
function upon exposure to bodily fluids within the
sub-retinal area, or for its susceptibility to elimination
upon exposure to prescribed conditions.
An additional factor to consider in determining
the make-up of compositions for support of the monolayer of
RPE cells is whether Bruch° s membrane has been or will be
removed from the host eye prior to transplantation of the
monolayer of RPE cells. Bruch's membrane serves to anchor
the RPE cells in a healthy eye. Removal of Bruch's
membrane may occur in cases of AMD where a subretinal
neovascular membrane has formed and Bruch's membrane is
removed as a consequence of the removal of the neovascular
membrane.
In cases where Bruch's membrane is removed, the
support composition will comprise a layer of collagen less
than 100 microns in thickness. and preferably between 1 and
10 microns in thickness. The basal surfaces of the RPE
cells attach readily to the collagen layer, which serves to
anchor the RPE cells to the choroid in place of the removed
Bruch's membrane. The layer of collagen also serves to
inhibit the occurrence or reoccurrence of subretinal
neovascularization through and around the transplanted
RPE. Such a collagen layer is retained indefinitely in the
94/25569 PCT/US93/04245
13
sub-retinal area. However, it is known that Bruch's
' membrane is essentially comprised of collagen, and such
microthin layers of collagen are permeable enough to avoid
impeding normal eye tissue functions such as the metabolic
exchange between the choroid and the retina. However,
collagen compositions of such a minimal thickness are not
strong enough to prevent buckling or distortion of the
monolayer of RPE cells during transplantation. Thus, such
microthin collagen materials need to be used in combination
with additional supporting materials which will be
eliminated after transplantation, if they are to form part
of the support for the transplanted RPE cells.
If transplantation is carried out with the native
Bruch's membrane intact. the host's RPE cells covering the
area to receive the transplant of RPE cells may be
physically and/or chemically debrided from the Bruch's
membrane, for example, by applying collagenase to the
native Bruch' s membrane to loosen the host' s RPE cells in
order to permit their removal. The transplanted RPE cells
may then anchor themselves directly to the Bruch's membrane.
Either to supplement a layer of collagen, as
discussed above, or in cases where inclusion of a collagen
anchor is not required, a support composition may be
selected which is dissipated, for ezample, by exposure to a
sufficient amount of heat, selected enzymes. or bodily
fluids. Gelatin is an eaample of a preferred support
material which is flexible, lacks toxicity to neural tissue
and has the ability to dissolve at body temperature. The
support composition may also comprise a material such as
activated low gelling temperature agarose (Type VII, #A
4018 Sigma Chemical Co., St. Louis) or fibrin, which are
known to dissipate when exposed to selective enzymes which
will not attack other compositions. For ezample, agarase
is an enzyme known to specifically attack agarose, and
WO 94!25569 PCT/US93/04245
14
urokinase is an enzyme specific to fibrin. Another
alternative is to use biodegradable polymers such as
ethylene-vinyl acetate copolymer (Elvax 40W, DuPont
Chemical Co., Delaware); poly(glycolic) acid,
poly(L-lactide-co-glycolide)(70:30 ratio) or poly(L- or
DL-lactic) acids (low mol. wt.) (Polysciences, Inc.,
Warrington, PA., Data Sheet #365, 1990). which are
flexible, non-toxic materials that slowly dissipate upon
implantation and exposure to bodily fluids. See, e.g.,
Powell, E.M., Brain Research, 515 (1990) 309-311 and
references cited in Polysciences, Inc. Data Sheet #365.
Advantageously, the gelatin or other support
composition may additionally serve as a carrier for any of
a number of trophic factors such as pharmacologic agents
including immunosuppressants such as cyclosporin A,
anti-inflammation agents such as deaamethasone,
anti-angiogenic factors, anti-glial agents and anti-mitotic
factors. Upon dissolution of the support composition, the
factor or agent becomes available to impart the desired
effect upon the surrounding tissue. The dosage can be
determined by established experimental techniques.
With appropriate enzymatic digestion, using
techniques disclosed by Pfeffer, B.A., Chapter 10,
"Improved Methodology for Cell Culture of Human and Monkey
Retinal Pigment Epithelium", prnarPSS in Retinal Research,
Vol. 10 (1991), at p.264, RPE cells are removed from the
Bruch's membrane as intact sheets rather than as
dissociated cells. Such a freshly harvested intact
monolayer of RPE cells may be immediately apposed as an
intact monolayer to a support, such as a thin collagen
sheet, supplemented with an overlayer or substrate of
gelatin, allowing several hours for adhesion prior to
transplantation. This procedure may be useful for
transplantation of RPE cells obtained in a biopsy. provided
94!25569 ~ PCTIUS93104245
.:
sufficient RPE tissue is obtained from the biopsy as an
' intact monolayer.
In most cases, however, it is preferable to
culture the harvested RPE cells on an attachment substrate
5 so that a monolayer of RPE cells may be prepared.
Culturing the RPE cells before harvesting is also preferred
both to allow for the production of larger populations of
RPE cells from a small amount of harvested tissue and to
allow for a period of observation to ensure that the RPE
10 cells to be transplanted are healthy and functional.
For proper culturing, harvested RPE cells
contained are apposed to a substrate to which they will
attach and grow, and which is capable of being maintained
in culture conditions appropriate for efficient growth of
15 RPE cells. Apposition of RPE cells may be accomplished by
pipetting a solution containing a, population of RPE cells
onto the substrate. Intact sheets of harvested RPE cells
in a physiological solution may also be physically released
via a wide-bore pipette onto the substrate and manipulated
with a fine camel-hair brush if necessary. Since RPE cell
cultures grow best at around 37° C, appropriate substrates
are solids at this temperature. Attachment substrates
which are suitable for growth of RPE cell cultures include
plastic culture ware or glass culture chamber slides coated
with collagen , laminin or fibronectin for cell
attachment. Coatings such as collagen may be applied to a
substratum such as fibrin or activated agarose.
Culturing harvested RPE cells can be accomplished
by any suitable method for obtaining a confluent monolayer
of RPE cells. Appropriate culturing techniques are
described in Pfeffer, B.A., Chapter 10, "Improved
Methodology for Cell Culture of Human and Monkey Retinal
Pigment Epithelium", Prog!ress in Retinal Research, Vol. 10
(1991); Ed. by Osborn, N. and Chader, J., or Mayerson,
WO 94/25569 PCT/LTS93/04245
~~.~16~~
16
P.L., et al., "An Improved Method for Isolation and Culture
of Rat Retinal Pigment Epithelial Cells", Investigative
Qphthalmologv & Visual Science, Nov., 1985, 2_C: 1599-1609.
Fig. 2 depicts a schematic of the preparation and
transplantation of a preferred embodiment of the implant
according to the invention. An implant laminate 1 is
formed as depicted in Figs. 2a and 2b. Gelatin 3 is
preferred for use to provide monolayer support during
transplantation because it is flezible, non-toxic to neural
tissues, and because it dissolves away after exposure to
body temperature (37° C). However, since RPE grows too
slowly at temperatures below 37° C, gelatin is not an
appropriate substrate for use in culturing RPE cells.
Thus, a monolayer of RPE cells 5 is first cultured on a
suitable substrate for growth. RPE cells will grow on
plastic culture ware A. However, to aid in removal of the
monolayer of RPE cells 5 from the culture ware A, the
culture ware A may be coated with a substrate 9 for the RPE
cells, such as agarose or fibrin. See Fig. 2b. The
substrate 9 is in turn coated with a thin (@ 2-4 microns)
layer of collagen 7, to which the monolayer of RPE cells 5
readily attach, and which will serve as an anchor for the
RPE cells in the post-transplantation period. If agarose
is used as the substrate 9, the agarose is activated so
that the collagen 7 will adhere to it by treating the
surface of the agarose with an activator such as cyanogen
bromide, as disclosed by Aaen, R. et al., N r (1967)
214: 1302-1304, or p-nitrophenyl chloroformate as disclosed
by Wilchek, M. and Miron, T., Eiochemistrv International
(1982) 4: 629-635. .
After a satisfactory culture of RPE cells has
been grown, a 5 to 30°s solution of molten gelatin is
applied to the apical surface of the monolayer of RPE cells
5 in the culture. The culture ware containing the RPE
~J 94/25569 ~ PCT/US93/04245
17
cells and gelatin solution is then cooled so that the
gelatin 3 is solidified into a sheet of preferably about
150-250 mm in thickness, attached to the apical surface of
- the RPE cells 5. The implant laminate 1 comprising a
monolayer of RPE cells 5, attached to the collagen anchor 7
and an overlayer of gelatin 3, may then be physically
removed from the culture ware A by slicing between the
culture ware A surface and the collagen 7 with a razor C,
as depicted in Fig. 2a.
The implant laminate 1 may also be removed from
the culture ware and substrate 9 enzymatically, as depicted
in Fig. 2b. The underlying substrate 9 may be removed from
the implant laminate 1 by application of an enzyme specific
to the material comprising the substrate 9. If the
substrate is agarose. agarase, an enzyme specific to
agarose, may be used to dissipate the agarose. A
concentration of 1 mg agarase/5 ml of culture medium will
suffice to dissipate the agarose substrate. If the
substrate consists of a fibrin coating, enzymes such as
urokinase (2 activity units urokinase/ml of culture media)
may be used to break apart the fibrin. To aid in
dissipation of the substrate 9, the culture ware A may
comprise a cell culture insert membrane, containing an
inner membrane A-1 with a porous bottom, and an outer,
solid membrane A-2. An ezample of such an insert membrane
is the Falconm Cyclopore"' membrane. The porous (e. g., pore
size of .45 mm) inner membrane A-1 is coated with the
substrate 9 during growth of the monolayer of RPE cells 5.
These pores facilitate penetration of the solution B
containing the enzyme into the substrate 9 so that it may
be broken apart, and the implant laminate 1 removed from
the culture ware A.
As depicted in Fig. 2c, the implant laminate 1 is
cut to create an implant la sized so that it will fit into
WO 94/25569 PCT/LTS93/04245
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a surgical instrument 20 which contains a carrier channel
for protection of the implant la during the transplantation
procedure. The implant la is transplanted to the
subretinal area at the posterior pole 76 of the host eye -
after detachment of the retina, as portrayed in Fig. 2d,
using surgical techniques more fully disclosed below. The
gelatin overlayer dissolves within hours after insertion of
the implant la into the host eye. After transplantation,
the retina reattaches, and the monolayer of RPE cells,
anchored to the layer of collagen, is sandwiched between
the choroid and the retina. See Fig. 2e.
Fig. 3 depicts a schematic of an alternative
embodiment of the implant according to the invention. As
described in relationship to Fig. 2 above. a monolayer of
RPE cells 5 is grown on culture ware coated with a
substrate 9. However, in this embodiment, the substrate 9
remains laminated to and comprises part of the implant la
during transplantation. The substrate 9 used for
transplantation may comprise agarose. which is biologically
inert and as discussed previously, may be dissipated by
exposure to agarase, an enzyme which selectively attacks
agarose. In this embodiment, activated agarose is coated
with a thin layer of collagen 7, about 2-4 microns in
thickness, which binds strongly to the activated agarose.
A monolayer of RPE cells 5 is then apposed to the collagen
7 and attaches readily to form a laminate of the RPE cells
5 and the support composition (collagen 7 and substrate 9)
to be used as an implant la for transplantation. Rather
than adding an overlayer of gelatin as discussed in
reference to Fig. 2, a razor is used to slice a sheet of
the material comprising the substrate 9 about 150-250 mm
thick to serve as a stabilizing support for the monolayer
of RPE cells 5 and the collagen 7 during transplantation.
94/25569 PCT/US93/04245
6.~ 6:~'~
19
Once transplantation is completed and the
monolayer of RPE cells has been stabilized in its proper
position, an intraocular injection of an enzyme such as
agarase (1 mg/5 ml vol. of vitreal chamber) if an agarose
substrate is used may be administered to break down the
substrate. Alternatively, the enzyme may be incorporated
into a slow-release agent 11 such as ethylene-vinyl acetate
copolymer (Elvax 40W, DuPont Chemical Co., Del.), which is
added to the substrate 9 prior to transplantation. Methods
for using biodegradable polymers as slow-release matrices
for enzymes are known in the art. such as disclosed by
Powell, E.M., Brain Research (1990) x:309-311; Wise, et
al.; Chapter 12, "Lactic/Glycolic Acid Polymers." Drua
Carriers in Biology and Medicine (Ed., Gregoriades), 1979:
Kitchell, J. and Wise, D., "Poly(lactic/glycolic acid)
Biodegradable Drug - Polymer Matrix Systems," Methods in
Enzymology, 112:436-448 (1985). Slow release of the enzyme
causes gradual break up of the substrate support. The
collagen 7 remaining after dissipation of the substrate 9
is physiologically permeable to and does not impede normal
tissue function, and serves to anchor the monolayer of RPE
cells 5, as well as to inhibit neovascularization in the
subretinal area of the host eye.
Fibrin, discussed in connection with Fig. 2, may
also be used as the substrate 9 in the embodiment depicted
in Fig. 3. Fibrin is the fibrous, insoluble protein that
forms the structural component for blood clots. Fibrin may
be pressed to add tensile strength and is a material which
provides a flexible, non-toxic support to which RPE cells
will attach and grow as a monolayer. To prepare fibrin,
fibrin clot suitable for substrate use is made by
catalyzing the polymerization of fibrinogen into fibrin by
the addition of thrombin. After transplantation, fibrin
will dissipate upon exposure to naturally produced enzymes
WO 94/25569 . ~ 1 PCT/US93/04245
which dissolve blood clots. If the dissipation of the
fibrin support composition is to be accelerated, enzymes -
which are specific for fibrin, such as tissue plasminogen
activator (TPA), urokinase or streptokinase may be .
5 administered using one of the methods for application of
enzymes discussed above.
To transplant the implant, the host eye is
prepared so as to reduce bleeding and surgical trauma. A
transcleral/transchoroidal surgical approach to the
10 subretinal space is an eaample of a suitable approach and
it will be understood that other surgical approaches, such
as a transcorneal approach, may also be used. The
preferred surgical approach in the human, Fig. 4, includes
making a transverse incision 70 in a sclera and choroid 78
15 of sufficient size so as to allow insertion of a surgical
instrument 20. The instrument 20 is advanced through the
sclera and choroid 78 and to the ora serrata 74 as
illustrated in Fig. 4. The instrument detaches the retina
as it is advanced under the retina and into the sub-retinal
20 space to the posterior pole 76 of the eye.
Preferably, an instrument comprising an elongate
tube having a flat, wide cross-section may be used so that
the implant may be drawn into the elongate tube for
protection as it is transported through an appropriate
sized incision in the sclera or choroid. The instrument is
advanced to the ora serrata 74 of the host eye and if the
instrument includes a lumen, the retina is detached by the
gentle force of a perfusate such as a saline-like fluid,
carboaymethylcellulose, or 1-2$ hyluronic acid ejected from
the lumen. Advantageously, the fluid may additionally
contain anti-oxidants, anti-inflammation agents,
anesthetics or agents that slow the metabolic demand of the
host retina.
If the instrument does not include a lumen, the
retina is detached by subretinal irrigation or by the walls
94/25569 PCT/US93/04245
21
of the surgical instrument as it is advanced under the
retina and into the subretinal space to the posterior pole
76 of the eye. The implant is then transplanted by
. retracting the tube containing the implant from the eye
while simultaneously gently ejecting the implant from the
tube. The instrument is then carefully withdrawn out of
the eye. Retinal reattachment occurs rapidly and the
monolayer of RPE cells is held in place in a sandwich-like
arrangement between the retina and the choroid. The
incision may require suturing.
Fig. 5 depicts a transcorneal surgical approach
as an alternative to the transscleral and choroidal
approaches described above. Except for the point of entry,
the surgical technique is essentially the same as outlined
for the transscleral or choroidal approaches. A transverse
incision 70 is made in a cornea 72 and the instrument 20
containing the implant is advanced under the iris, through
the cornea 72 and to the ora serrata 74 as illustrated in
Fig. 5. The iris should be dilated for example, with
topical atropine. The edges of the corneal incision are
abutted and sutured if necessary to allow healing. The
transcorneal approach is preferred for rodents because it
has been found to reduce bleeding and surgical trauma.
Nevertheless, a transscleral or choroidal approach is
preferred for humans to avoid scarring of the cornea which
may interfere with visual acuity.
A further surgical approach is to diathermize in
the pars planna region to eliminate bleeding. The sclera
is then incised and the choroidal and any native epithelial
tissue is diathermized. The surgical tool is then inserted
through the incision, the retina is intercepted at the ora
serrata and the implant is deposited in the subretinal area
otherwise as outlined elsewhere herein.
In yet a further surgical approach, entry is
gained through the pays planna area as outlined above and
WO 94/25569 PCT/US93/04245
22
an incision is made in the retina adj acent to the retinal
macula. The surgical tool is then inserted through the
retinotomy and into the macular area.
As discussed previously in connection with the
make up of the support compositions used in accordance with
the invention, after transplantation, the implant is
exposed to a predetermined set of conditions, such as
ezposure to heat, selected enzymes or bodily fluids. Upon
the appropriate amount of exposure, the support composition
is dissipated or will not otherwise impede normal eye
tissue function of the host eye and the transplanted
population of RPE cells.
The following ezamples illustrates the invention.
EXAMPLE 1
E~cper~mental Animal and Materials
RPE cells were taken from the sub-retinal area of
donated human eyes (obtained from the Missouri Lions and
St. Louis Eye Banks) following corneal removal. Hosts were
adult albino rats immune-suppressed with cyclosporin A 10
mg/kg/day IP injection).
Harvesting of RPE Cells
Immediately after corneal removal, the eye was
pinned by the optic nerve stump into an eye cage and placed
into an eye jar. The jar was flooded to capacity with cold
Delbecco's modified essential medium (DMEM), supplemented
with 5% fetal bovine serum. The eye was processed in a
sterile environment. After loose connective tissue and
muscle were carefully trimmed from the eye, it was placed
upright on a sterile plate and the anterior segment with
the adherent vitreous was lifted out of the eye cup. Four
slits were cut radially toward the optic disc with the eye
lying flat in a petri dish. The neural retina was then
4
94/25569 PCT/US93/042 5
23
separated at the optic disc and removed. The shell was
' then washed with Hanks' balanced salt solution, Ca++ and
Mg++ free, and treated with 0.25% trypsin for 30 min. at
- 37° C. The trypsin solution was aspirated from the shell
and DMEM (GIBCO) was added. The cells were released from
Bruch's membrane by gently brushing the surface of the RPE
cells with the polished tip of a pasteur pipette and
pipetting the culture medium in the shell.
Culturing' of RPE Cells
Glass culture chamber slides were prepared by
coating them with liquid collagen obtained from Sigma
Chemical Co., St. Louis. The collagen was allowed to
solidify to about 150 microns in thickness. 3X105 RPE
cells were plated onto the prepared slides using a pasteur
pipette. The RPE cells were then incubated in DMEM + F12
(1:1. GIBCO) supplemented with 20% fetal bovine serum
(Sigma Chemical Co., St. Louis). The culture medium was
changed every 3-4 days until a confluent monolayer of RPE
cells had been cultivated. Implants comprising monolayers
of RPE cells laminated to collagen were removed from the
culture chamber slides and sheets of approximately 2 mm X 4
mm were cut out for transplantation.
Burctical Procedure
A transverse incision was made in the cornea
sufficient to allow insertion of a surgical instrument that
is 2.5 mm wide with a lumen 0.5 mm high. The instrument
was advanced under the iris (dilated with topical atropine)
to the ora serrata, detaching the retina. The carrier was
then advanced under the retina into the subretinal space to
the posterior pole of the eye. The instrument allowed an
implant comprising a monolayer of RPE cells apposed to a
150 mm collagen substrate (up to 2.5 X 4 mm) to be guided
WO 94/25569 PCT/US93/04245
24
into the retinal space by advancement of the plunger with
simultaneous retraction of the surgical instrument. The
instrument was then removed. Following removal of the
instrument, the edges of the corneal incision were abutted
to allow rapid, sutureless healing. The eye was patched
during recovery and a prophylactic dose of penicillin was
administered. Upon removal of the patch, a veterinary
ophthalmic antibiotic ointment was applied.
Transplant recipients were maintained on a 12
hr/12 hr light/dark cycle with an average light intensity
of 50 luz. Following appropriate survival times, the
animal was overdosed with pentobarbital.
Fig. 6 shows a view of the transplanted monolayer
of RPE cells positioned under the retina, with the cornea,
pupil and lens removed, two weeks post-transplantation.
The arrows designate the edges of the implant.
Histologic Preparation
For histologic evaluation, rat eyes with RPE cell
implants were immersion-fixed in Bouin's solution for 5
hrs., then dehydrated in a graded ethanol series. The eyes
were trimmed with a razor blade to include the transplanted
area for implanted retinas, then processed through aylene
and embedded in paraffin. Sections were cut at 8mm and
stained with hematoxylin and eosin.
Results
By using the transcorneal approach, it was found
that the positioning of the monolayer of RPE cells between
the host's retina and the adjacent choroidal tissue layer
of the eye could be accomplished while minimizing the
vascular damage and subsequent bleeding into the eye. In
addition, it was found that this approach does not appear
to disrupt the .integrity of the retina, which reattaches to
~l 94/25569 PCT/US93104245
the back of the eye with the transplanted RPE cells
interposed between the retina and the choroid. Using this
insertion method, it was possible to position the RPE cells
beneath the posterior pole of the retina (Fig. 7).
5 To determine the viability of the transplanted
RPE cells, paraffin sections (8 mm) were made from the eye
receiving the RPE cell transplant at 2 weeks, 4 weeks, and
3 months after transplantation. It was found that the
monolayer of RPE cells survived transplantation at all
10 times tested (10 out of 12 transplants).
With immune-suppression, successful transplants
were seen at all survival times so far ezamined (2 weeks to
3 months), showing apparent physical integration with the
host eye and maintaining morphological features of the RPE
15 as illustrated in Fig. 7 which shows a transplant of a
moriolayer of human RPE cells from adult donor to adult rat
host. (T, transplant). 40X.
Paraffin sections made at 2 weeks post-
transplantation are shown in Figs. 7 and 8. Fig. 7 is a
20 low-power photomicrograph showing the location of the RPE
monolayer transplant (T) between arrowheads at the
posterior pole of th host eye. Note the maintenance of
normal retinal configuration as well as the normal
appearance of the RPE cells, maintained in substantially a
25 monolayer between the choroid and the outer segments of the
photoreceptor cells. Figure 8 is a higher-power
photomicrograph showing in detail the interface between the
transplant and the adjacent retina. As shown in Fig. 8,
the photoreceptor outer segments are shown to be in normal
apposition to the apical microvillar processes (A) of
transplanted RPE cells.
The functional capabilities of the transplanted
RPE cells and reconstructed retina were ascertained by
their maintenance of photoreceptor bodies, and the presence
WO 94/25569 PCT/ITS93/04245
m=
" ~~ 26
of inner and outer segments with proper orientation to the
apical processes of transplanted RPE cells. The success of
these procedures for transplanting monolayers of RPE cells
are reflected in Figs. 7 and 8.
'.EXAMPLE 2
As a comparison, mature human RPE cells were
transplanted in dissociated form using the bolus injection
method as set forth by Li and Turner in Eag. Eye Res.,
47:911 (1988). RPE cells from the same donor tissue used
for the transplants conducted as set forth in Eaample 1
were harvested and cultured as set forth in Example 1.
After culturing, the RPE cells were trypsonized to form
dissociated RPE cells for transplantation by bolus
injection according to the method of Li and Turner. Hosts
were adult albino rats immune-suppressed with cyclosporin A
10 mg/kg/day IP injection) as set forth in Example 1.
Transplant recipients were maintained on a 12 ".
hr/12 hr light/dark cycle with an average light intensity
of 50 lua. Following appropriate survival times, the
animal was overdosed with pentobarbital. Paraffin sections
of the eye receiving the RPE cell implant were then cut (8
mm) .
Paraffin sections made at 2 weeks post-
transplantation are shown in Figs. 9 and 10. Fig. 9 is a
low-power photomicrograph showing the location of the
injection of dissociated RPE cells at the posterior pole of
the host eye. Note the pathological configurations,
including retinal detachment (RD), retinal pucker (RP),
subretinal fibrosis (SF) and retinal rosette (RR)
formation. Figure 10 is a higher-power photomicrograph
showing in detail the pathological invasion of RPE cells
into the adjacent retina, subretinal fibrosis and retinal
pucker.
94/25569 PCT/US93/04245
27
From the foregoing description those skilled in
the art will appreciate that all aspects of the present
invention are realized. The present invention provides an
improved surgical implant that is adapted to provide cell
organization during transplantation of the RPE cells. With
the implant of this invention cell organization is
maintained during and after RPE transplantation.
In view of the above, it will be seen that the
several objects of the invention are achieved and other
advantages attained.
As various changes could be made in the above
compositions and methods without departing from the scope
of the invention, it is intended that all matter contained
in the above description or shown in the accompanying
drawings shall be interpreted as illustrative and not in a
limiting sense.
