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W Tao, P Stabila, B Dean, W Bell; Construction, Characterization and Safety Testing of CNTF Secreting Mammalian Cell Lines for Encapsulated Cell Therapy . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2312.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The objective of the current study was to construct, characterize, and safety evaluate the CNTF secreting cell lines that will be used for encapsulated cell therapy for treating retinal degenerative diseases. Methods: Two stable CNTF secreting human mammalian cell lines were established by transfecting human RPE cells with a CNTF gene containing plasmids. The CNTF secreting cell lines were designated NTC-201-10 and NTC-201-6A. The growth characteristics, morphology and CNTF production were monitored for over one year in culture. Their encapsulated performance was evaluated both in vitro and in vivo: CNTF production was quantified by ELISA, and bioactivity and specificity of CNTF were evaluated in a TF-1 CN5a.1 cell bioassay. Viral safety and tumorigenecity of the cell lines were also examined. Results: Both NTC-201-10 and NTC-201-6A cell lines maintained normal RPE morphology with stable CNTF output in vitro for over a year. Un-encapsulated NTC-201-10 cell line produced ∼200 ng/106 cells/day, and NTC-201-6A cell line produced ∼800 ng/106 cells/day. Cell-secreted CNTF was biologically active, with specific activity 2∼3 fold higher than that recombinant CNTF. Furthermore, the observed bioactivity was completely blocked by a goat anti-CNTF neutralizing antibody. When encapsulated, both cell lines secreted efficacious levels of CNTF in vivo, which was quantified upon device explant at 1∼5 ng/day for NTC-201-10 and 10∼20 ng/day for NTC-201-6A, respectively. The cell lines tested negative for viruses and are not tumorigenic in nude mice. Conclusion: Two stable CNTF secreting cell lines were successfully established which maintained normal growth characteristics and morphology for over one year in vitro, and produced biologically active CNTF. Their encapsulated performance and safety profile make them ideal for Encapsulated cell therapy.
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