U Hartmann1, F Sistani, U H Steinhorst. 1. Augenklinik der Medizinischen Hochschule Hannover, Carl-Neuberg-Strasse 1, D-30625 Hanover, Germany.
Abstract
PURPOSE: To establish a method for transplantation of cultured monolayers of RPE and IPE into the subretinal space, anterior lens capsule was evaluated for its suitability to serve as growth support and carrier for transplantation procedures. MATERIALS AND METHODS: Twenty-four anterior lens capsules were obtained from porcine eyes. The same number of human lens capsules was obtained during cataract surgery. Six lens capsules of each species were stored at -80 degrees C. Subsequently, the capsules were transferred onto type-I collagen. A second set of six lens capsules was treated identically except for the cryo treatment. A third set of six capsules was initially exposed to 0.05% trypsin for 30 min. Suspended porcine RPE and IPE cells (5 x 10(4) cells/well) were seeded on the top of each capsule. The remaining six lens capsules served as controls and were incubated in uncoated 12-well dishes without undergoing experimental treatment. The cultures were maintained in a water-saturated atmosphere at 37 degrees C with 5% CO(2). Six days later, viability, morphology, and cell density were determined. The capsules covered by a confluent monolayer of cells were transferred into uncoated wells and cultivated for another 10 days. At the end of the experiment, light and phase-contrast microscopy was performed on all capsules. RESULTS: Storage at -80 degrees C and exposure to trypsin resulted in significant reduction of cellular contamination. The highest cell density was found after 5 days when capsules which had undergone cryopreservation or trypsin exposure served as support for RPE and IPE. The pigment cell layer was firmly attached to the capsules and permitted a transfer to other culture flasks without significant cell loss. The IPE cell layer remained confluent after transfer to uncoated culture flasks, while the RPE cell layer ceased to proliferate 10 days after transfer. CONCLUSIONS: Lens capsules may be suitable for growing and supporting monolayers of pigment epithelial cells. Especially IPE cells formed stable monolayers on anterior lens capsules which could be transferred to secondary culture flasks without inflicting damage on the cells.
PURPOSE: To establish a method for transplantation of cultured monolayers of RPE and IPE into the subretinal space, anterior lens capsule was evaluated for its suitability to serve as growth support and carrier for transplantation procedures. MATERIALS AND METHODS: Twenty-four anterior lens capsules were obtained from porcine eyes. The same number of human lens capsules was obtained during cataract surgery. Six lens capsules of each species were stored at -80 degrees C. Subsequently, the capsules were transferred onto type-I collagen. A second set of six lens capsules was treated identically except for the cryo treatment. A third set of six capsules was initially exposed to 0.05% trypsin for 30 min. Suspended porcine RPE and IPE cells (5 x 10(4) cells/well) were seeded on the top of each capsule. The remaining six lens capsules served as controls and were incubated in uncoated 12-well dishes without undergoing experimental treatment. The cultures were maintained in a water-saturated atmosphere at 37 degrees C with 5% CO(2). Six days later, viability, morphology, and cell density were determined. The capsules covered by a confluent monolayer of cells were transferred into uncoated wells and cultivated for another 10 days. At the end of the experiment, light and phase-contrast microscopy was performed on all capsules. RESULTS: Storage at -80 degrees C and exposure to trypsin resulted in significant reduction of cellular contamination. The highest cell density was found after 5 days when capsules which had undergone cryopreservation or trypsin exposure served as support for RPE and IPE. The pigment cell layer was firmly attached to the capsules and permitted a transfer to other culture flasks without significant cell loss. The IPE cell layer remained confluent after transfer to uncoated culture flasks, while the RPE cell layer ceased to proliferate 10 days after transfer. CONCLUSIONS: Lens capsules may be suitable for growing and supporting monolayers of pigment epithelial cells. Especially IPE cells formed stable monolayers on anterior lens capsules which could be transferred to secondary culture flasks without inflicting damage on the cells.