Long Term Performance and Viability of Genetically Engineered Retinal Pigment Epithelial (RPE) Cells Secreting CNTF Encapsulated in Hollow Fiber Membrane Devices

Abstract

Abstract: : Purpose: The performance and viability of genetically engineered RPE cells encapsulated in hollow fiber membrane devices, termed NT–501, were investigated over a 9 week culture period. Methods: Thirty six devices were manufactured under standard procedures and incubated at 37°C. Starting at 2 weeks post manufacture, 4 devices were removed each week for 9 weeks and assessed for performance and viability by CNTF secretion, lactate dehydrogenase (LDH) release, histology, and TUNEL analysis. Devices treated with mitomycin C served as positive controls for cell death. CNTF secretion was assessed using a commercial ELISA kit (R&D Systems). LDH release (cell membrane integrity) was assessed using the CytoTox–ONE Homogeneous Membrane Integrity Assay (Promega). Apoptosis was monitored by the incorporation of fluorescein 12–dUTP using the Dead End Fluorometric TUNEL Assay (Promega). Histological examination of the devices was performed using standard hematoxylin and eosin staining techniques. Results: The CNTF secretion of the encapsulated cells was consistent with historical data over the 9 week period. The cell membranes of the encapsulated cells demonstrated high levels of integrity as only small amounts of LDH release could be detected from each device. Histological examination of the devices showed that the majority of the cells from each test device were healthy during the 9 week period. Only a small percentage of cells from devices analyzed at each time point appeared to be picnotic or unhealthy. TUNEL analysis showed that a small percentage of cells in each test device analyzed over the 9 weeks contained nuclei with fragmented DNA. The majority of the cells failed to incorporate the fluorescent 12–dUTP label indicating that the bulk of cells were healthy, a finding which was consistent over the 9 week period. No fluorescence was visualized in cells which did not receive the enzyme mix (negative control). Four devices treated with 5µm and 10µm mytomycin C showed dose dependent decreases in CNTF release, increases in apoptosis by TUNEL staining, and increases in the number of darkly staining picnotic cells by hematoxylin and eosin staining. Conclusions: The results of this study suggest that the performance and viability of the encapsulated cells are stable, as consistent CNTF levels were released and only a small amount of cell death was observed beyond the existing shelf life. No significant differences in viability were observed among devices analyzed between 2 and 9 weeks using these methods.

Keywords: growth factors/growth factor receptors • retinitis • photoreceptors