In Vitro Evaluation of Polymer Encapsulated CNTF Secreting Mammalian Cell Lines Over the Course of a Ten Month Period

Abstract

Abstract: : Purpose: To develop encapsulated cell technology (ECT) for ophthalmic applications, we investigated the shelf life of encapsulated genetically modified CNTF secreting human mammalian cells (NTC-201) over the course of ten months. Previous studies indicated the good performance of polymer encapsulated cells placed in closed packages containing culture media and held for up to two months in vitro followed by a one month implantation in the rabbit vitreous. Extending the shelf-life of the ECT product in a closed package will facilitate development and clinical evaluation of ECT. Methods: Two genetically modified human mammalian cell lines (NTC-201-10 and NTC-201-6A) secreting low and high levels of CNTF were encapsulated within polymer membrane devices and placed in closed holding packages containing 40 ml of culture media. The package units were incubated at 37^oC. Devices were tested for CNTF output using a commercially available ELISA kit and sacrificed for histomorphology at 2 week, 1, 2, 3, 4, 6, and 10-month time points. Encapsulated cells at the 10 month time point were enzymatically dissociated from the device scaffolding, grown to confluence and analyzed by phase contrast microscopy, stained and analyzed using the fluorescent probes calcien and ethidium and investigated for RPE specific CRALBP and RPE65 markers using RT-PCR. Results: Both high and low producing encapsulated cells show relative stability of each cell line over the course of the 10-month in vitro hold period. CNTF levels relative to the two cell lines are statistically different between 2 weeks and 4-months (p < 0.001) but begin to converge at the 6-month time point (p = 0.652) as the pH of the culture media begins to decline. Comparison between unencapsulated and 10 month encapsulated cells show similar morphologic characteristics by fluorescent and phase contrast analysis. RPE65 and CRALBP protein levels were detected. Conclusions: Previously, encapsulated cells were maintained for up to two months with no negative impact on in vivo performance. The data from this study suggests that further development of the maintenance conditions could extend the shelf-life of the ECT product for up to 10 months. Compared to unencapsulated cells, no morphological or phenotypical changes were identified in the encapsulated cells maintained for ten months.

Keywords: transplantation • retina: distal(photoreceptors, horizontal cell • retinal pigment epithelium