Long Term in vitro Stability of CNTF Secreting NT-501 Implants

Abstract

Purpose: : To investigate the long term in vitro stability of CNTF secreting NT-501 implants.

Methods: : NT-501 implants were manufactured using standard protocols and held at 37°C in closed packages containing 37 mls Endo-SFM for 12 weeks. Implants were pulsed for CNTF release in 1 ml of Endo-SFM for 24 hours at 2, 8, 10, and 12 weeks post manufacture. Cell containing implants were analyzed for metabolic activity and then subjected to either total DNA or histological analysis. CNTF release from implants was quantified using a commercial ELISA kit (R&D Systems). Cell metabolism was determined using the CCK-8 assay (Dojindo). Total DNA was determined using the Hoefer DyNA Quant 200 fluorometer. Histological examination of the implants was performed using standard hematoxylin and eosin staining techniques.

Results: : In vitro analysis of high CNTF producing implants showed a stable release of CNTF from 2 to 12 weeks. High producing implants released between 20 and 24 ng CNTF/device/24 hours during the 12 week testing period. Analysis also showed that the encapsulated cells were metabolically active with CCK-8 readings of 0.700 and 1.000 (OD₄₅₀) during the 12 week testing period. Analysis of total device DNA showed that the high CNTF producing implants maintained a stable cell mass over the testing period with implants containing average of 250,000 to 290,000 cells over the 12 week period. Low CNTF producing implants released between 5 and 8 ng CNTF/device/24 hours during the 12 week testing period. Analysis also showed that the encapsulated cells were metabolically active with CCK-8 readings between 0.600 and 1.000 (OD₄₅₀) during the 12 week testing period. Analysis of total device DNA showed that the low CNTF producing implants maintained a stable cell mass over the testing period with implants containing an average of 230,000 to 310,000 cells over the 12 week period. Histological examination of both the high and low CNTF producing implants showed that the majority of the cells from each device were healthy. Very few cells from either the high or low CNTF producing implants appeared to be picnotic or unhealthy during the 12 week testing period.

Conclusions: : Both high and low CNTF secreting NT-501 implants maintain stable cell mass and function over a 12 week period suggesting a minimal shelf life of 3 months for this cell based therapy.

Keywords: retina • growth factors/growth factor receptors • apoptosis/cell death