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C.G. Thanos, B. Dean, K. Kauper, W. Tao; Microcarrier Cell Culture for Precise Dose Regulation and Control of Cell Proliferation in the NT–501 Intraocular Device. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):5045.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:The NT–501 intraocular device for the treatment of retinitis pigmentosa has been established as an effective mode of CNTF administration to the retina and can provide therapy on the order of years. Two cell lines identical in phenotype but differing in CNTF secretion profiles have been used to achieve variable doses, and altering the internal device volume can provide further dose regulation. The PET scaffold in the NT–501 devices provides variable proliferation. The current study evaluates Cytodex® and Cultispher® microcarriers on their ability to provide more controlled and predictable dose regulation and cell proliferation within the device. Methods:Cytodex®1 and 3, microcarriers approximately 200 µm in diameter with only their external surface available for attachment, and Cultisphere® S and G, roughly the same size with an available internal porous matrix, were cultured with NT–201–10 (low CNTF producer) and NT–201–6A (high producer) in spinner flasks for up to 2 weeks at 37°C and subsequently encapsulated at varying densities into modified NT–501 devices. CNTF output, viability, and cell attachment were monitored for 2, 4, 8, or 12 weeks in vitro in the standard NT–501 packages containing 40 mL endothelial serum–free media (ENDO–SFM). Following the incubation period, CNTF production was analyzed by incubating devices in fresh ENDO–SFM for 24 hours followed by detection with an ELISA. Viability, proliferation, and attachment were assessed using immunocytochemistry with confocal microscopy and scanning electron microscopy (SEM). Results: All scaffold materials successfully accommodated cell attachment. Confocal analysis revealed >95% viability on confluent Cytodex® microcarriers after 5 days with both cell lines in microcarrier suspension culture, whereas Cultispher® microcarriers took roughly 10 days for confluence with comparable viability. Fluorescent staining revealed focal points of attachment with cells appearing fibroblast–like in morphology on Cultispher® microcarriers and cobblestone–like on Cytodex® microcarriers. Encapsulated microcarriers sustained CNTF secretion for the duration of the experiment and distinct dosing ranges were achieved. Both Cultispher® types with either cell line showed statistically significant dose increases over their Cytodex® counterparts based on total microcarrier number. Conclusions:Microcarrier cell culture is a feasible and possibly advantageous method of encapsulation in the NT–501 device. By controlling cell density and proliferation at the subculture level, a more efficient and controlled pharmacologic profile can be achieved.
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