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C. McGovern, S. Sherman, P. Stabila, J. Lydon, A. Lee, B. Dean, K. Kauper, D. Litvak, W. Tente, W. Tao; Characterization of CNTF Release From ECT Devices With Long–Term Incubations . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5124.
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© ARVO (1962-2015); The Authors (2016-present)
The objective of this study was to examine CNTF release dynamics of in vitro devices that had extended incubation times using brefeldin A (BFA), a well characterized reversible inhibitor of constitutive secretion.
NT–201–6A cells engineered to secrete CNTF were encapsulated into devices composed of hollow fiber membrane with either a relatively high diffusive flux (NT–50X) or intermediate flux (NT–501). Devices were held at 37° C from three weeks (young) to fifty two weeks (old) in complete media. After the hold period, devices were pulsed for 24 hours in complete media (pre–BFA phase), then pulsed in media supplemented with BFA (inhibitory phase), followed by a final pulse in BFA–free media (recovery phase). At the conclusion of the study, devices were processed for histology to assess morphology and viability. CNTF levels were quantitated via ELISA kit (R&D Systems). Histological examination was performed using hematoxylin and eosin (H&E) staining.
For each device inhibition and recovery outputs were expressed relative to pre–BFA outputs, which were defined as 100%. NT–50X devices held one year, as well as NT–501 devices held 3 weeks, showed a precipitous decrease in CNTF in response to BFA and a significant recovery of CNTF levels post–drug, similar to unencapsulated cells. Older NT–501 devices demonstrated a reduction similar to that of control untreated devices, which remained virtually unchanged in the recovery phase. Histologically the more open devices (NT–50X) held for 1 year had very good viability, as did all young NT–501 devices (treated and control). One year NT–501 devices contained approximately 10–20% viable cells and significant cellular debris. Pre–drug output on all devices was consistent with historical data.
NT–50X devices demonstrated very good viability and CNTF output out to one year, and was responsive to BFA treatment, indicating excellent de novo CNTF synthesis. NT–50X may be a promising candidate for future ECT development by significantly extending product shelf life.
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