Abstract
Purpose:
To investigate the feasibility of cryopreservation as a potential means of Encapsulated Cell Technology (ECT) device storage after manufacturing.
Methods:
An NTC-200 cell line secreting an antiangiogenic factor was used for the encapsulation. Cells were expanded in a growth media, harvested, and formulated at a specific density in a serum free media, either with or without various concentrations of a cryopreservative. Prepared cells were loaded into Neurotech ophthalmic ECT devices. Control devices were kept at 37C while cryopreserved devices were frozen at a rate of 1C/min and stored in liquid nitrogen (vapor phase) for 2 weeks. Cryopreserved devices were then removed from liquid nitrogen, rapidly thawed, washed in a balanced salt solution, and incubated in a serum free media for 6 days. The devices were then cultured in fresh serum free media for 24 hours. The condition media samples were collected and assayed for the antiangiogenic factor by ELISA. The devices were subsequently analyzed for metabolic activity using the CCK-8 assay (Dojindo) and then subjected to either total DNA using the Hoefer DyNA Quant 200 fluorometer (Pharmacia) or histological analysis using standard hematoxylin and eosin staining techniques.
Results:
During the pre-encapsulation culture period, the cells maintained a typical and consistent cuboidal morphology. Cells encapsulated in devices in the fresh media group and cryopreserved group both maintained a normal morphology with a high density of healthy cells distributed throughout the device during the testing period. No deterioration of the device capsule was observed by visual inspection. Factor secretion levels, cell metabolic activity, and the overall device cell load between fresh and cryopreserved devices were also comparable. Furthermore, secreted proteins with expected molecular weights were detected in the culture media from both device groups. No differences in protein size or degradation between the fresh and cryopreserved device groups were detected by Western blot.
Conclusions:
These data suggested that the cryopreservation is a feasible method of extending the shelf life of ECT products. Longer term and in vivo studies of cryopreserved devices are currently under investigation. Optimizing cryopreservation techniques in the manufacturing process will allow for the long term storage of ECT product and may greatly enhance accessibility of ECT products worldwide.
Keywords: 412 age-related macular degeneration •
609 neovascularization •
701 retinal pigment epithelium