Amniotic membrane as supportfor human retinal pigmentepithelium (RPE) cell growth

Carmen Capeans,1,2 Antonio Pineiro,2 Marıa Pardo,2 Catalina

Sueiro-Lopez,3 Marıa Jose Blanco,1 Fernando Domınguez4 and

Manuel Sanchez-Salorio2

1Servicio de Oftalmologıa, Complejo Hospitalario Universitario de Santiago, Santiago

de Compostela, Spain2Instituto Gallego de Oftalmologıa, Santiago de Compostela, Spain3Departamento de Biologıa Fundamental, Facultad de Biologıa, Universidad de

Santiago de Compostela, Santiago de Compostela, Spain4Departamento de Fisiologıa, Facultad de Medicina, Universidad de Santiago de

Compostela, Santiago de Compostela, Spain

ABSTRACT.

Purpose: The aim of this work was to culture human retinal pigment epithelium(hRPE) cells over human amniotic membrane (hAM). Human AM was studied

for its viability as an adequate support for transplantation of an hRPE cell

monolayer with preserved cell polarity to the subretinal space.

Methods: Human AM was obtained from pregnant women during caesareansection. The hAM was sectioned and the pieces were fixed to culture dishes.

Human RPE cells were cultured from adult corneal donors and were seeded over

hAM. Phase-contrast photographs were obtained. Selected specimens were pro-

cessed by transmission electronic microscopy (TEM).

Results: The attachment and growth of hRPE cells over hAM was observed.Human RPE cells constituted tight colonies that maintained epithelial phenotype.

Using TEM, we identified a monolayer of hRPE cells, with cuboidal to spher-

oidal morphology. These cells showed integration with the substrate and cell�cellcontacts were detected.

Conclusion: Amniotic membrane may be a suitable substrate for hRPE growth.Further studies are required in order to determine the viability of hRPE on hAM

in the subretinal space.

Key words: amniotic membrane – cell culture – retinal pigment epithelium – retinal transplantation

Acta Ophthalmol. Scand. 2003: 81: 271–277Copyright # Acta Ophthalmol Scand 2003. ISSN 1395-3907

Introduction

Retinal cell transplantation is anexperimental therapeutic approach tocertain degenerative diseases of theretina (i.e. age-related macular degen-eration (AMD) and retinitis pigmen-

tosa). Different procedures have beenstudied in experimental and clinical stud-ies, with varying degrees of success:implantation of fetal or adult humanretinal epithelial cells into subretinalspace (Algvere et al. 1994), and trans-plantation of autologous iris pigment

epithelial (IPE) cells (Crafoord et al.2002) and cultured photoreceptors(Kaplan et al. 1997). Retinal pigmentepithelium (RPE) transplantation hasbeen shown to rescue photoreceptorcells in dystrophic rats (Lin et al. 1996).Retinal pigment epithelium cell

transplantation has been attemptedin patients with non-exudative andexudative AMD by means of severaltechniques (cell suspension, patchtransplants) (Algvere et al. 1999). Themain disadvantage of cell suspensiontransplantation is the random organ-ization in multilayers of cells in the sub-retinal space of the host (Crafoord et al.1999). Other complications have beendescribed, such as subretinal fibrosis,invasion of the retina by pigmentedcells and retinal pigment epithelialcells in the vitreous cavity causing pro-liferative vitreoretinopathy (Liu et al.1992).In order to preserve cell orientation,

some researchers have tried to maintaincell monolayers and polarity by meansof different supports. These includebiological supports such as Descemet’smembrane (Thumann et al. 1997), lenscapsule (Hartmann et al. 1999), Bruch’smembrane or blood cryoprecipitates(Farrokh-Siar et al. 1999). Othergroups have studied the application of

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synthetic supports, such as collagensubstrates (Bhatt et al. 1994), bio-degradable polymer films (Lu et al.1998) or microspheres (Oganesani et al.1996).The human amniotic membrane

(hAM) is a thin and elastic tissue thatforms the inner layer of the amnioticsack. Human AM, with its thick base-ment membrane and avascular stromalmatrix, has been successfully used forsurface reconstruction in a variety ofocular surface disorders (severe pter-igyum, chemical burn, ocular cicatricialpemphygoid and Stevens–Johnson syn-drome) (Tseng et al. 1997). In thesecases, hAM works as an optimal bio-logical support for conjunctival cellgrowth. Recent publications show thathAM constitutes an adequate substratefor in vitro growth and expansion ofconjunctival epithelial progenitor cells(Grueterich & Tseng 2002; Meller et al.2002). Shimazaki et al. (2002) reportedshort-term clinical results after trans-plantation of human limbal epitheliumcultivated on amniotic membranefor the treatment of severe oculardisorders.Rosenfeld et al. (1999) used a rabbit

model to demonstrate that subretinalimplantation of hAM appears to bewell tolerated without evidence ofinflammation.In view of all these considerations,

the purpose of the present work wasto study the amniotic matrix as a feas-ible substrate for the attachment andgrowth of hRPE cells.

Material and Methods

Human amniotic membrane (hAM)

Two hAMs were obtained from twopregnant women during caesarean sec-tion after obtaining informed consent.Under sterile conditions, the hAM

was processed as described previouslyby Tseng et al. (1997) for hAM trans-plantation to ocular surface disease.Briefly, the hAM was washed withDulbecco’sModification ofEagle’sMin-imal Essential Medium (DMEM, GibcoBRL, Gaithersburg, Maryland, USA),supplemented with 100U/ml penicillin,100mg/ml streptomycin (ICN Biomed-icals, Costa Mesa, California, USA)and 2.5mg/ml amphotericin B (Sigma-Aldrich Quımica, Madrid, Spain). Inthe present study the hAM was not

frozen, but was freshly used in allexperiments.

Human retinal pigment epithelial (hRPE)

cultures

Human RPE cultures were establishedfrom adult human corneal donors tothe Eye Bank of the Complejo Hospi-talario Universitario de Santiago. Oneeye of each corneal donor (42 and 30-year-old men) was utilized for hRPEculture.The culture technique followed that

of previous reports (Capeans et al.1998). Briefly, under sterile conditions,the anterior segment, vitreous body andretina were discarded. The eyecup wasfilled with DMEM supplemented with10% fetal calf serum (FCS) (GibcoBRL, Gaithersburg, Maryland, USA)and 0.25% trypsin-0.02% EDTA(Sigma-Aldrich Quımica, Madrid,Spain). It was then incubated at 37� in5% CO2 humidified atmosphere for45mins. After this, hRPE cells wereremoved by gentle pippetting. ThehRPE cells obtained were transferred to60mm culture dishes (Nunc, Roskilde,Denmark) in a complete mediumcomposed of 10% FCS, 100U/ml peni-cillin, 100mg/ml streptomycin, 2mM

L-glutamine (ICN Biomedicals, CA,USA) and 2.5 mg/ml amphotericin B inDMEM. In their initial passages, thecells had a polygonal pigmented mor-phology and they grew forming mono-layers. To check that the cells had anepithelial origin, they were stained forcytokeratin (AE1/AE3) as previouslydescribed (Leschey et al. 1990). ThehRPE cells used throughout this workwere taken between the second and fifthpassages and were always culturedunder subconfluence conditions.

NIH3T3 fibroblasts cultures

NIH3T3 fibroblasts were cultured incomplete medium composed by 10%FCS, 100U/ml penicillin and 100mg/mlstreptomycin in DMEM on plasticdishes (Nunc, Roskilde, Denmark).

Preparation of hAM with hRPE cells

In order to remove the attached amni-otic epithelial cells, huge pieces of hAMwere incubated in dispase II (1.2U/mlDispase II; Roche Diagnostics, Molecu-lar Biochemicals, Barcelona, Spain) inCa2þ Mg2þ free phosphate-bufferedsaline (PBS) for 15mins, at room tem-perature under sterile conditions.

Afterward, the hAM pieces werewashed in PBS. Thus, the epithelialhAM layer could be easily and gentlyremoved. Phase-contrast microscopycontrols were carried out in order toensure that the hAM was free of epithe-lium. The hAM pieces were then sec-tioned into 2� 2 cm fragments using asurgical blade. These fragments wereattached to glass slides by means ofcyanocrylate adhesive (Histoacryl, BBraun, Tuttlingen, Germany) with theepithelial basement membrane facingup. The metallic guide was removedfrom a 25-gauge Abbocath-1-T (AbbottIreland Ltd, Dublin, Ireland) andthe device was used to connect theplastic tube to a Histoacryl bottle inorder to obtain a single, small drop ofcyanocrylate. The adhesive was appliedcarefully, keeping the hAM as tense aspossible. Once the adhesive was dry,the glass slides were placed into empty90mm culture dishes (Nunc, Roskilde,Denmark).Human RPE cells from subconfluent

second to fifth passages were tryp-sinized and counted. A total of 1.5� 106cells were seeded by gentle pippettingover each piece of hAM attached tothe glass slides. In the control casesthe same number of NIH3T3 cellswere seeded over hAM. Each culturewas properly identified. In order toreach cell�hAM attachment, culturedishes were carefully filled with com-plete medium 30mins after cell seeding.The culture media was changed every3days and daily phase-contrast controlswere performed. Microphotographs weretaken with a phase-contrast microscopeduring the culture period. Some of theseimages were enlarged in the photogra-phy laboratory in order to improve theidentification of details.

Transmission electronic microscopy

Selected specimens were processed fortransmission electronic microscopy(TEM) after 30 days in culture.Human AM with hRPE or NIH3T3fibroblasts was fixed with 2% glutar-aldehyde (Sigma-Aldrich Quımica,Madrid, Spain) in 0.1M phosphate buf-fer pH7.3 for 2 hours. After fixation,the hAM sections were postfixed in1% OsO4 in 0.1M phosphate buffer for15mins at room temperature, dehydratedin a graded series of alcohols andembedded in Spurr. Semi-thin sections(1mm thick) were cut on a Reichert-Jung

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ultramicrotome (Ultracut-E) and thenstained with toluidine blue and studiedunder a light microscope. Finally, ultrathin sections (50–80Zm thick) werestained with uranyl acetate and leadcitrate for 5mins and observed andphotographed with a Philips CM-12Transmission Electron Microscope(TEM) (SEI, ElectronOptics, Eindhoren,the Netherlands). Some TEM imageswere enlarged in the laboratory in orderto improve the identification of details.

Results

Morphology and viability studies

Under phase-contrast microscopy,freshly obtained hAM showed anepithelial surface constituted by acobblestone epithelium. Isolated pig-mented epithelial cells were found in allstudied specimens (Fig. 1A). Dispase-treated hAM showed its epithelial

basement membrane face after gentlescraping (Fig. 1B). In our study, thehAM thickness ranged between 90 and190mm. The hAM fixed by means ofcyanoacrylate maintained its adherenceto the glass slides for at least 4weekswithout detaching.

Cell cultures over hAM

The human RPE cells cultured overdispase-treated hAM fragments wereattached to the epithelial basementmembrane side within 24 hours of seed-ing. After settling down, hRPE cellsdisplayed their typical in vitro morph-ology. Human RPE cells were organizedin tight colonies constituted by small,polygonal and uniform epithelial cells,after 3–5 days of culture (Fig. 1C, D).No differences were found in morph-ology, attachment and proliferation ofhRPE cells from the two culturesutilized. Control NIH3T3 cells seededover hAM took a similar period of time

until adhesion, showing a spindle-shaped morphology and an irregulararrangement over hAM (data notshown).

Transmission electronic microscopy

When hRPE cells were seeded overdispase-treated hAM, semi-thin sectionsdemonstrated that, after 30 days of cul-ture, the cells were organized in a tightmonolayer of large cuboidal to roundcells (Fig. 2). Consistent with this, theTEM study showed a tight monolayerof cuboidal to spheroidal hRPE cellsgrowing over epithelium-free hAM(Figs 3 and 4). The hAM matrix wasconstituted by collagen spindles orga-nized in different directions andembedded in an amorphous and clearground substance (Fig. 3, blackarrows). We found well-defined foot-plates (Fig. 3, FP), observed as elonga-tions of hRPE cell basal membraneimmersed in hAM. It was easy to find

Fig. 1. Phase-contrast microphotographs. (A) Cobblestone disposition of the amniotic epithelium in a freshly obtained hAM. Sporadic pigmented

epithelial amniotic cells were found (white arrows). [Original magnification �40.] (B) After dispase treatment basement membrane was exposed.[Original magnification �40.] (C, D) After seeding and culture of hRPE cells over denuded hAM, tight colonies (black arrowheads) could be

identified. In both cases, pictures were taken after 30 days in culture. [Original magnification: (C) �40; (D) �60.]

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intercellular membranes runningparallel from apical to basal poles(Fig. 3, black arrowheads). No desmo-somes were detected; nevertheless, wefound some areas characterized byapposition of electrodense membranes(Figs 3 and 4). Moreover, several focalcontacts, separated by cleftlike spaces,were detected. Prominent and isolatedbuds (Fig. 3B) were identified at the api-cal side of hRPE cells.Cellular components such as mito-

chondrias and endoplasmic reticulumwere present (Fig. 3, MI and R, respect-ively). Melanin granules (melano-somes), characteristic of these types ofcells, were easily identified (Fig. 3, ME).Large-sized nuclei were balloon-shapedand were placed in the centre of thecuboidal cells (Figs 3 and 4, N). Chro-matin was dispersed and sporadicnuclear pores were seen.Nevertheless, NIH3T3 cells, as seen

under optical microscopy, were smallerthan hRPE cells and showed an irregu-lar arrangement (Fig. 5). By means ofTEM, we proved that these cells grewin a multilayer disposition. Finally, nocell�cell or cell�hAM attachmentscould be seen in these cells.

Discussion

Alteration in the RPE layer occurs in abroad spectrum of inherited and/or

acquired retinal degenerative disorders(loss of RPE cells results in photorecep-tor dysfunction in non-exudative andexudative AMD). The surgical removalof RPE cells during excision of subret-inal neovascularization leads to atro-phy of the underlying choriocapillaris,limiting visual recovery. The spectrumof diseases that can potentially betreated by RPE transplantation hasbeen expanded by findings that describemutations in the RPE65 gene as theorigin of autosomal recessive childhood-onset severe retinal dystrophy (Gu et al.1997). The idea of replacing agedand/or diseased RPE with healthyRPE grafts has been extensively inves-tigated in animal models and inhumans (Algvere et al. 1994; Radtkeet al. 2002; Tsukahara et al. 2002). Ret-inal pigment epithelium and iris pig-ment epithelial transplants have beenshown to survive well in the subretinalspace (Crafoord et al. 2002) andprevent photoreceptor degeneration tosome extent (Lin et al. 1996). It seemsthat transplantation in the form of apatch is important in preventing celldisorganization, excessive mechanicalstress or damage of the well orientedcells and in facilitating precise localiza-tion of the graft. Several supportmatrices have been utilized in differentstudies (Bhatt et al. 1994; Oganesaniet al. 1996; Thumann et al. 1997; Luet al. 1998; Farrokh-Siar et al. 1999;

Hartmann et al. 1999; Tezel & DelPriore 1999; Tsukahara et al. 2002).Most of these supports turned outeither to be toxic to the cells or to dis-integrate on exposure to liquid media.Human amniotic membrane consists

of a single cell layer of epitheliumbound to a continuous basement mem-brane, which is constituted of type IVcollagen and laminine, and which inter-faces with an avascular collagenousstroma composed of interstitial col-lagen and elastin. Human AM is anelastic and transparent tissue that iseasy to obtain from pregnant women:there is normally a great amount ofhAM available during a caesarean sec-tion. Procedures for handling and stor-ing hAM have been well described byother groups (Tseng et al. 1997).It has been demonstrated that trach-

eal epithelium cultured on amnioticmembrane showed attachment anddifferentiation (Noguchi et al. 1995).Recent findings indicate that humancorneal epithelial cells can be success-fully regenerated in vitro over amnioticmembrane by a limbal epithelium cell-suspension culture system (Koizumiet al. 2002).In this paper, we propose the use of

epithelium-free hAM as support forhuman RPE growth. Rosenfeld et al.(1999) showed that it is feasible toimplant hAM into the rabbit subretinalspace and showed how hAM can bewell tolerated by the rabbit retina.Tsukahara et al. (2002) demonstratedthat freshly harvested human RPEcells seeded over Bruch’s membraneexplants show better attachment if thebasement membrane is not debrided.These results suggest that RPE celltransplantation requires a basementmembrane in order to ensure cell attach-ment.We have demonstrated that hRPE

cells seeded over hAM showed adhe-sion to the hAM in about 24 hours.Moreover, hRPE cells maintain epithe-lial features (morphology, pigment)and can proliferate over epithelium-free hAM. The hRPE cells growingover hAM were found to be highlyorganized. These cells constitute a tightmonolayer with well defined cell�celland cell–substrate interactions. As acontrol for the hRPE cells, we usedNIH3T3 transformed fibroblasts seededover hAM. Conversely, these cells wererandomly organized in multilayerswithout apparent cell–cell interactions.

Fig. 2. Toluidine blue stained sections of: (A) hRPE and (B) NIH3T3 cells after 30 days in culture

over hAM (AM). Black arrows point to seeded cells in both cases. Scale bars: (A) 208mm;(B) 150mm.

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The development of cell polarity overhAM has been described previously byNoguchi et al. (1995), who worked withtracheal epithelium. Some authors havereported the existence of tight junctionsbetween RPE cells after a period ofculture over substrate (Hartmann et al.1999). We believe these findings dependon the biological function assumed bycells over a new environment – notethat the neurosensory retina, chorio-capillaris and choroid are not presentin in vitro systems. Although we foundcertain cell structural polarity in ourmodel, we do not know whetherhRPE, seeded over hAM or other sup-ports, can organize itself into a physicaland functional barrier. It will be neces-sary to carry out additional assays inorder to study the degree of functionalrelationship between hRPE cells andhAM (i.e. rod outer segments degrada-tion studies).

Several in vivo human studies havedemonstrated that hAM is an optimalsubstrate for conjunctival growth insevere damaged ocular surfaces; it hasbeen observed that hAM develops tightintegration with superficial ocular tis-sues (conjunctiva, cornea), promotingre-epitelization. The mechanisms bywhich this phenomenon occurs are stillunknown. However, it has been sug-gested that amniotic stroma can secretegrowth factors or express adhesionmolecules that can facilitate cellgrowth. We believe that these charac-teristics make hAM an adequatesupport to RPE growth and furthertransplantation. Thus, hAM couldplay an important role in maintainingthe viability and integration of RPEcells into the subretinal space.To our knowledge, Rosenfeld et al.

(1999) did not reduce the thickness ofhAM and they showed that the pres-

ence of hAM did not induce visiblesigns of rejection or outer retinal disor-ganization. The hAM utilized in ourwork had variable thickness, rangingbetween approximately 90 mm and190mm, and we did not select hAMfragments according to thickness.Further experiments, carried out in ani-mals, could require a diminution ofhAM thickness in order to maintainthe retinal architecture and promoteRPE cell integration. We believe thiscould be reached by means of enzym-atic digestion of the hAM matrix (i.e.collagenase).Despite the fact that the eye is immuno-

logically privileged, immune rejectionmay be a barrier to successful retinaltransplantation. Our work was devel-oped in vitro. We believe that furtherexperiments must focus on rejectionof hAM implanted to subretinalspace. For this, animal models

Fig. 3. Transmission electron microphotograph (photomontage) of hRPE after 30 days of culture over epithelium-denuded hAM. Amniotic

membrane matrix (AM) shows collagen spindles organized in different directions (black arrows) and embedded in an amorphous and clear

fundamental substance. Human RPE cells were organized in a tight monolayer of large cuboidal to round cells. Note intercellular membranes running

parallel from apical to basal poles (black arrowheads). Apposition of electrodense membranes is seen. We found well-defined footplates (FP),

observed as elongations of hRPE cell basal membrane immersed in hAM. Isolated and prominent buds (B) were detected in the apical side. Cellular

components mitochondria (MI) and endoplasmic reticulum (R) were found. Melanin granules (melanosomes), characteristic of this kind of cells, were

easily identified (ME). Large-sized nuclei were balloon-shaped and were placed in the centre of the cuboidal cells (N). Chromatin was dispersed and

sporadic nuclear pores were seen. AS: apical side; BS: basal side. Scale bar: 3mm.

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would be needed to determine rejectionlevels (autologous versus allotrans-plants). One hypothetical idea arisingfrom our work concerns the possibility

of amniotic membrane antigenicimmunity.Our work suggests that epithelium-

free hAM might be an optimal sub-

strate for RPE transplantation to thesubretinal space. Further experimentsmust be carried out to establish thedegree of integration of this graftwithin the host retina (extensive animalstudies) and analyse the molecularproperties of RPE cells on this sub-strate.

Acknowledgements

This work was supported by a grant bythe Galician Education Department toManuel Sanchez-Salorio (grant no. XUGA90201B97).Authors’ thanks are due to Professor

Tomas Garcıa-Caballero (Departamentode Anatomıa Patologica, Complejo Hospi-talario Universitario de Santiago deCompostela) for his collaboration inimmunocytochemical techniques for cyto-keratin staining of hRPE cells. The authorsalso thank Professor Celina Rodicio Rodicioand Professor Isabel Rodrıguez-Moldes

Fig. 4. (A) TEM microphotography shows an area of contact between hRPE cells after 30 days of culture over hAM. No hemidesmosomes could be

detected, but several areas of membrane contact were found (arrowhead). N: nucleus. (B) TEM microphotography magnifies the membrane contacts

shown in (A) (arrowheads). N: nucleus. (C) TEM microphotography shows multiple focal contacts between membranes of hRPE cells cultured over

hAM (arrowheads). N: nucleus. (D) High magnification in TEM microphotography shows an intimate apposition between hRPE membranes and

condensation of cytoplasmic and intermembranous spaces (black arrow). Scale bars: (A and C) 3mm; (B and D) 1mm.

Fig. 5. NIH3T3 cells as seen in optical microscopy were smaller than hRPE cells and showed an

irregular arrangement. By means of TEM, we proved that these cells (F) grew in a multilayer

disposition. No cell�cell or cell�hAM attachment could be seen. AM: hAM. Scale bar: 2 mm.

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(Departamento de Biologıa Fundamental,Universidad de Santiago de Compostela)for their collaboration in the selection andstudy of TEM images. Finally, the authorsthank Miro Barreiro Perez (Servicio deMicroscopıa Electronica, Universidad deSantiago de Compostela) for technical assist-ance and Andrew Day for help in the trans-lation of the text.

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Received on 11 September, 2002.

Accepted on 13 February, 2003.

Correspondence:

Dr Antonio Pineiro Ces

Instituto Gallego de Oftalmologia

Hospital de Conxo

Rua Ramon Baltar s/n

15706 Santiago de Compostela

Spain

Tel:þ 34 981 534 501Fax:þ 34 981 531 627Email: mjbtapc@corevia.com

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