Eye (1989) 3, 27-32

Transplantation of Retinal Pigment Epithelium U sing a Pars Plana Approach

C. LANE, M. BOULTON and J. MARSHALL London

Summary We describe a pars plana approach for transplantation of autologous retinal pigment epithelial cells from a peripheral chorioretinal biopsy to prepared Bruch's membrane at the posterior pole of the same eye. The technique has potential application in research and could be clinically applicable in the treatment of senile macular degeneration.

Tissue transplantation is an expanding medi­ closed system.6 RPE cell attachment and cal field in which corneal grafting has tradi­ focal recovery of overlying retina was tionally been the ophthalmic surgeon's prime demonstrated. However, neither model was interest. However recent successes using directly applicable to the clinical situation. neuronal cells, such as substantia nigra Use of the open sky technique with a retinal homografts1 and adrenal medullary autog­ flap prevented reattachment of undamaged rrafts2•3 in the treatment of Parkinson's dis­ retina in the monkey model and the rabbit ease, have stimulated further research into eye had limitations as a model of the human retinal transplants. Such transplants have eye. Firstly, there are few retinal blood ves­ potential use in the treatment of a variety of sels and secondly, rabbit RPE cells have retinal dystrophies and degenerations affect­ large lipid globules which may affect their ing the photoreceptors and the retinal pig­ dynamics. ment epithelium (RPE). Both the RPE and In order to use a model which had closer photoreceptors are of neuroectodermal ori­ similarity to the human eye, we chose to gin and are accessible for transplantation in assess the technical feasibility of RPE trans­ the mature human retina. We have chosen to plantation in the pig eye. This has a very study transplantation of RPE because it is the similar retinal structure and blood supply to simpler tissue with which to establish a the human eye, although there is no macula technique. In addition it is easily visualised or fovea.7,8,9 The miniature pig was chosen because of pigmentation and mature cells can for ease of long term follow-up and autolog­ readily be grown in tissue culture prior to ous grafts were used to avoid tissue rejection. transplantation.4 Cells from primary cultures were chosen to RPE transplantation in two animal species optimise cellular morphology and metabolic pas previously been researched by Gouras. function, which become less consistent in In the first cultured human RPE cells were subcultures, 10 injected onto Bruch's membrane in an owl monkey using an open sky technique.s A Method severe inflammatory reaction, suggestive of Retinal Detachment flcute rejection,. ensued. In the rabbit Access to the RPE was achieved using a pars homologous cells were injected through a plana approach. A localised retinal detach­ micropipette into the subretinal space using a ment of 3 to 5 disc diameters was produced

Correspondence to: Dr Mike Boulton, Institute of Ophthalmology, Judd Street, London wei 28 C. L. LANE ET AL by steady injection of a jet of 0.05 ml of Dul­ viously.15 Pure primary cultures of RPE cells becco's phosphate buffered saline without in microtitre plates were radio labelled using calcium and magnesium (PBSA) against the 0.2 uCilml of methyl-3H-thymidine,5 added inner retina through the 30 gauge port of a to the medium one week prior to the esti­ fibreoptic diathermy tissue manipulatorll mated time of reaching confluence. Cell cul­ (Fig. 1). This is similar to the technique ini­ ture was necessary to amplify cell numbers, tially described in rabbits.12 In the miniature but primary cultures were chosen to optimise pig retinal reattachment spontaneously cellular morphology and metabolic func­ occurred within 2-5 days. 13 tion.lO In the most recent series a second Preliminary transplantation experiments label of a cell suspension of carbon particles were performed using homologous cells and was added to the medium 24 hours prior to the final series of eight experiments was per­ confluence. formed using autologous cells from a chorioretinal biopsy. Transplantation The recipient site was prepared at the post­ Preparation of Donor Cells erior pole of the same eye. Bruch's mem­ A chorioretinal biopsy was obtained using a brane was denuded to avoid apex to apex technique similar to that described by orientation of donor to host RPE. This has Peyman.14 A scleral flap was retracted and been reported by Anderson in the catl6 and isobutyl cyanoacrylate glue (Histacryl, observed by ourselves in localised areas of Cyanamid) was applied directly to the retinal detachment in the miniature pig and exposed choroid. Using the hardened glue prevents satisfactory function of overlying for fixation, a 3.5 mm square of choroid, photoreceptors. To debride a localised area a RPE and retina was excised using Vanna's retinal detachment was induced and the port scissors and placed in culture medium at of the manipulator was immediately room temperature this consisted of MEM, advanced through the retinotomy. Injection supplemented with 20% fetal calf serum. The of a jet of 0.05 ml PBSA against the RPE medium was cooled to 4°C for transport to resulted in a localised area of debridement, 1 the laboratory. Cortical vitreous was abscised to 3 mm in diameter. Immediately after prep­ and the scleral wound was repaired using 8/0 aration of the recipient site the manipulator ethilon. Subconjunctival injections of 2 mg was withdrawn. A suspension of lOO!!1 of betamethasone and 20 mg gentamicin were trypsinised RPE cells, containing only given. approximately 4000 cells in order to avoid RPE cells were isolated and grown in tis­ multilayers, was injected into the subretinal sue culture from the biopsy as described pre- space using a 27' gauge needle. The sclerotomy was repaired and eyes were fixed in 4% glutaraldehyde and processed for light microscopy and autoradiographyS at 1 hour (3 eyes), 4 hours (2 eyes), 4 days, 7 days and 6 months after transplantation.

Results Three principal aspects of this technique for transplantation of small numbers of autolog­ ous cells in the miniature pig eye revealed significant problems. Firstly, although the process of retinal detachment was usually atraumatic, reattached retina did not com­ pletely return to normal (Fig. 2). Secondly, preparation of the recipient site resulted in Fig. 1. Pars plana approach for the induction of neat separation of host cells from Bruch's retinal detachment. membrane in most cases (Fig. 3), but the Fig. 2. Light micrograph of reattached retina two days after detachment. Note: vacuolation of the inner portion of the photoreceptor outer segments with some pyknotic nuclei and prominent apical microvilli on the RPE . Bar marker 50iJ11l.

Fig. 3. Scanning electron micrograph showing denuded area (D) of Bruch's membrane underlying retinal detachment. Bar marker200 ftm. 30 C. L. LANE ET AL denuded area was variable, frequently very Discussion near to the retinotomy site, and associated These results suggest that considerable prac­ with microscopic haemorrhage. Thirdly, tical difficulties exist with attempts to trans­ delivery of RPE cells into a pool of sub-reti­ plant RPE cells into the posterior pole using nal fluid was inconsistent. In a few cases closed intraocular microsurgery. To over­ transplanted cells, most readily recognised by come these it will be essential to refine the intra-cytoplasmic carbon granules, were instrumentation to reduce the risk of seen lying on Bruch's membrane (Fig. 4). haemorrhage and to mInImISe damage However, there was a tendency for trans­ caused to overlying retina by detachment. In planted cells to lie near the edge of the retinal addition it would be essential to optimise detachment, well away from the central delivery of RPE cells to the central debrided debrided area, and occasional cells were also area of Bruch's membrane. identified in the vitreous cavity and along the In a rat model fresh pigmented donor RPE inner limiting membrane. Although trans­ cells have recently been transplanted over planted cells on Bruch's membrane were intact host albino pigment epithelium with­ identifiable up to 4 hours, subretinal haemor­ out preparatory retinal detachment and with rhage and an inflammatory infiltrate formed focal morphological recovery of overlying a barrier, preventing attachment of donor photoreceptors.17 Because of the host's cells in two cases prepared 4 and 7 days after albinism this could be performed transcler­ transplantation. Donor cells have not been ally into the retinal periphery with adequate identified at sections from 6 months after visualisation. Although such a simple autologous transplantation. Hence, reattach­ technique may have reduced the degree of ment of healthy overlying photoreceptors has trauma to the overlying retina in the minia­ not yet been demonstrated in this model. ture pig, choroidal haemorrhage is more

Fig. 4. Electron micrograph of carbon (C) labelled RPE cell lying on Bruch's membrane 1 hour after trans plantation. Many red blood corpus cles (RB C) are seen. Bar marker5 [!m. TRANSPLANTA nON OF RETINAL PIGMENT EPITHELIUM 31 likely and visualisation of transplanted cells ant photoreceptor degeneration. The poten­ would not be possible in the pigmented ani­ tial use here would be minimal, but, if the mal. Moreover, an internal approach is pref­ technique could be applied to the transplan­ erable for access to the macula, where trans­ tation of photoreceptors, it could prove use­ plantation has greatest potential and ful in treating retinal dystrophies. Present avoidance of damage to Bruch's membrane research into transplantation of photorecep­ and the choriocapillaris is critical. However, tors is at a very early stage.23.24 the use of an external approach avoids depos­ RPE transplantation, using autologous ition of pigment epithelial cells in the vitre­ cells, could be applied to the treatment of ous cavity, an event which may predispose to macular degeneration. The occurrence of the development of proliferative vitreo­ disciform lesions is related to alterations in retinopathy. 18 Bruch's membrane and accumulation of Vitrectomy was not performed in this debris between the RPE and choroid with model because the retina reattached rapidly choroidal neovascularisation.25 Present treat­ with the vitreous in place. Even if vitrectomy ment using laser photocoagulation is destruc­ had been performed in this model, it is tive and has limited application.26 unlikely to have been complete. Pig vitreous Miller et al.27 have demonstrated that its is very thick and the cortical vitreous gel is effect may be related to the formation of firmly attached to the inner retina, so total tight junctions between proliferated RPE removal of this part of the vitreous without cells over a neovascular membrane, so the damage to the underlying retina would have transplantation of RPE cells into this area proved difficult. In the adult human eye could potentially seal off a neovascular com­ detachment of the posterior vitreous gel and plex. The peripheral retina is relatively pre­ vitrectomy are factors which could well served;26 healthy RPE cells from this area improve cell delivery to the subretinal space. may potentially be used as donor tissue. Two other elements may also increase the success rate in the human eye. Firstly, References Bruch's membrane in the adult is signific­ 1 Dunnet SB, Bjorklund A, Steveni U, Iversen antly thicker than in the young miniature pig SD: Grafts of embryonic substantia nigra rein­ and may be more resistant to trauma. Sec­ nervating the ventrolateral striatum amelior­ ondly the use of a site which has already been ate sensorineural impairments and akinesia in prepared by a disease process would avoid rats with 6-0HDA lesions of the nigrostriatal traumatic denudement of Bruch's mem­ pathway. BrainRes 1981, 229: 209-17. brane. If the technique is further refined to a 2 Madrazo I et al.: Open microsurgical autografts stage at which it could be used on the human of adrenal medulla to the right caudate nuc­ leus in two patients with intractable Parkin­ eye, when could it be applied? son's disease New EngJ Med1987,316:831-4 . Malfunction of the RPE has been impli­ 3 Morrison KE: Brain transplantation - still fan­ cated in certain retinal dystrophies. There tasy? Discussion paper. J Roy Soc Med 1987, are isolated reports of excess amounts of 80:441-4. lipofuscin and similar degradation products 4 Edwards RB: Culture of mammalian retinal in the sub-retinal space or RPE in Lawrence­ pigment epithelium and neural retina. In Moon-Biedl syndrome, 19 fundus flavi­ Methods in Enzymology Vol 81: Visual pig­ macuiatus20 and Best's disease.21 This is con­ ments and purple membranes, L. Packer ed., sistent with the appearance of a dark choroid New York Academic Press 1982: 39-43. 5 on fluorescein angiography and could reflect Gouras P, Flood MT, Kjeldbye H, Bilek MK, Eggers H: Transplantation of cultured retinal defective phagocytosis. Clinically an RPE epithelium to Bruch's membrane of the owl defect is suggested by a flat EOG in Best's monkey's eye. Curr Eye Res 1985, 3: 253-65. disease21 and early pigment loss in (0 Lopez R, Gouras P, Brittis M, KjeJdbye H: choroideremia22 These dystrophies do not Transplantation of cultured rabbit retinal severely affect central vision until adulthood epithelium to rabbit retina using a closed eye or later and RPE transplantation would have method. Inves t Ophthalmol Vis Sci 1986, 28: to be performed before the onset of signific- 1131-7. 32 C. L. LANE ET AL

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