Encapsulated Cell Technology for the Delivery of Biologics to the Mouse Eye

Summary

Presented here is a protocol for the use of alginate as a polymer in microencapsulation of immortalized cells for long-term delivery of biologics to rodent eyes.

Abstract

Many current therapeutics under development for diseases of the posterior pole of the eye are biologics. These drugs need to be administered frequently, typically via intravitreal injections. Encapsulated cells expressing the biologic of choice are becoming a tool for local protein production and release (e.g., via long-term drug delivery). In addition, encapsulation systems utilize permeable materials that allow diffusion of nutrients, waste, and therapeutic factors into and out of cells. This occurs while masking the cells from the host immune response, avoiding the need for suppression of the host immune system. This protocol describes the use of alginate as a polymer in microencapsulation coupled with the electrospray method as a microencapsulation technique. ARPE-19 cells, a spontaneously arising human RPE cell line, has been used in long-term cell therapy experiments due to its lifetime functionality, and it is used here for encapsulation and delivery of the capsules to mouse eyes. The manuscript summarizes the steps for cell microencapsulation, quality control, and ocular delivery.

Introduction

Cell-based therapies represent revolutionary biological techniques that have been applied widely in medicine. Recently, they have been successfully applied in the treatment of neurodegenerative diseases, eye diseases, and cancer. Cell therapies cover a wide range of fields from cell replacement to drug delivery, and this protocol focuses on the latter. Biodegradable alginate microcapsules (MC) have shown effectiveness as a delivery system, and they are becoming widely used in the biomedical field. Alginate has been used in microencapsulation due to its simple gelling process, biodegradability, excellent biocompatibility, and stability under in vivo conditions

Protocol

All experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Medical University of South Carolina Animal Care and Use Committee under protocol ID 00399.

1. Cell Culture

1. Generate human retinal pigment epithelial cells (ARPE-19) cell line stably expressing the gene of choice according to published protocols^14,15.
2. Maintain cells.......

Discussion

This cell encapsulation technique is relatively quick and easy to perform; however, certain points must be kept in mind to obtain accurate downstream results. Cells should be maintained in culture in a Petri dish prior to encapsulation and held at proper confluency. Encapsulation should be performed in a proper ventilation hood with regulated air flow, if possible. Too strong of an air current can affect capsule formation, especially in long-term experiments. Sterile utensils and solutions are critical for long-term main.......

Materials

Name	Company	Catalog Number	Comments
3 mL Syringe	BD	309656
30 G 1" Blunt needle	SAI Infusion technology	B30-100
Alginic acid sodium salt, from brown algae	Sigma	A0682
Atropine Sulfate Ophthalmolic solution (1%)	Akorn	NDC 17478-215-15	for pupil dilation
BD 1 mL Syringe 26 G x 3/8 (0.45 mm x 10 mm)	Becton, Dickinson and Company	DG518105 500029609 REF 309625	to generate the guide hole
Calcium chloride, Anhydrous, granular	Sigma	C1016
GenTeal Tears	Alcon	NDC 0078-0429-47	to lubricate the eyes during anesthesia
Goniotaire: Hypromellose (2.5%) Ophthalmolic Demulcent Solution (Sterile)	Altaire Pharmaceuticals Inc.	NDC 59390-182-13	to lubricate the eyes during anesthesia
Hamilton Needle/syringe Tip: 27 Gauge, Small Hub RN NDL, custum length (12mm), point style 3, 6/PK	Hamilton	7803-01	for intravitreal delivery of capsules
Hamilton Syringe: 2.5 µL, Model 62 RN SYR, NDL Sold Separately	Hamilton	7632-01	for intravitreal delivery of capsules
HEPES buffer, 1M	Fisher Bioreagents	BP299100
High voltage generator	ESD EMC Technology	ES813-D20
LIVE/DEAD Viability/Cytotoxicity Kit	Thermofisher Scientific	L3224
L-Ornithine hydrochloride, 99%	Alfa Aesar	A12111
Neomycin and Polymyxin B Sulfates and Dexamethasone Ophthalmolic Ointment	SANDOZ	NDC 61314-631-36	antibiotic to prevent infection after intravitreal injection
Phenolephrine Hydrochloride Ophthalmolic Solution (2.5%)	Akorn	NDC 17478-201-15	for pupil dilation
Sodium Chloride	Sigma	S-5886
Sterile syringe filters, 0.2 um	VWR	28143-312
Syringe pump	GRASEBY	MS16A