Optimization of Cell Encapsulation Methods to Target the Anterior Segment

Abstract

Purpose: : To optimize a method of delivering protein products to the anterior segment via cell encapsulation. This method has the advantage of using cells to secret biologically active agents while polymer encapsulation sequesters and protects them from immune rejection.

Methods: : Immortalized lens epithelial cells (B3) were transfected with a modified pIRIS plasmid expressing a bicistronic mRNA for two reporter genes including secreted-luciferase (Metridia longa) and EGFP (enhanced green fluorescent protein) driven by the CMV promoter. Levels of expression of the reporter gene were measured by evaluating luciferase activity in the culture media using the Ready-To-Glow ^TM Secreted Luciferase System (BD biosciences).The transfected B3 cells were encapsulated in alginate beads with the Air-Jet method, using a NISCO encapsulation unit J30. The size of the alginate beads and the number of cells per bead were optimized by controlling the density of cells in the alginate solution before encapsulation and the flow rates of N2 gas and cell suspension through the encapsulation unit. The rigidity of the alginate beads was controlled by the concentration of CaCl2 in the solution for bead incubation. Using this method it was possible to encapsulated cells in beads of sizes ranging from 50 to 200 µm. The beads contained cells expressing EGFP and released secreted luciferase. The encapsulated B3 cells were injected intracamerally in three rabbit eyes and intravitreally in two rabbit eyes. Controls received a sham injection of polymer only. Complete ocular examinations and intraocular pressure measurements were performed twice daily for five days. On day five, the rabbits were sacrificed and both aqueous humor and vitreous were collected. Luciferase activity was measured as outlined previously.

Results: : Statistically significant greater luciferase activity was found in the aqueous humor of all experimental eyes compared to background controls. The luciferase activity of anterior chamber aqueous specimens from both intracameral and intravitreal injections were relatively similar.

Conclusions: : Encapsulated cells placed intracamerally or intravitreally result in the successful delivery of proteins to the anterior chamber. This is potentially a new method of drug delivery for the treatment of disorders such as glaucoma.

Keywords: gene transfer/gene therapy • injection • anterior chamber