Development of an Optimized Culture Media to Improve VEGF Antagonist Secretion and Stability by Encapsulated Cell Technology Implants

Abstract

Purpose: : Encapsulated Cell Technology (ECT) is designed to deliver therapeutic factors directly to the retina through a semi-permeable, hollow fiber membrane implant that encapsulates genetically modified cells. The goal of this study was to optimize ECT pre-implant culture conditions to maximize and stabilize secretion of a VEGF-antagonist targeting microgram per day daily production levels and to remove all animal and human components from the culture media. Effects of culture media and additional supplementation on encapsulated cell protein secretion levels and encapsulated cell health were evaluated.

Methods: : ECT devices were manufactured and encapsulated using a platform cell line engineered to secrete a VEGF antagonist molecule. Media supplements, including lipids, vitamins, amino acids and growth factors were added to the platform culture media in an effort to increase protein expression and optimize encapsulated cell line performance. Multiple commercial media designed for enhanced recombinant protein expression, differentiation, and viability were compared to the current platform media. Protein production of the encapsulated cells was evaluated by ELISA. Device health and cell viability was analyzed qualitatively by the preparation of histology slides. Media analyte and metabolite concentration were evaluated using a chemistry analyzer.

Results: : The addition of GlutaMAX^TM and removal of L-glutamine from all media led to decreased ammonia accumulation and improved encapsulated cell stability. One animal and human component free media formulation increased protein production of the encapsulated cell line three to four fold, maintaining steady-state expression levels in micrograms per day. Most media supplements evaluated did not improve protein expression, with the exception of cholesterol. However, long-term exposure to high concentrations of cholesterol was detrimental to cell health. While select stem cell factors improved protein production, changes to cell morphology and growth rate occurred as well.

Conclusions: : Optimization of media used to support ECT function resulted in identification of an improved formulation capable of achieving sustained ECT production of a VEGF antagonist at a rate of micrograms daily secretion. In addition, the absence of animal and human components enables this media to be used for clinical development and commercialization.

Keywords: age-related macular degeneration • gene/expression • growth factors/growth factor receptors