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^1,2,3,4.

The electrospray method, as a microencapsulation technique, has been successfully utilized to encapsulate peptides and proteins using alginate (base polymer) and poly-l-ornithine (secondary coating polymer). Both polymers are naturally found and used for their biocompatibility^5,6,7. However, the main challenge in cell-based therapies is suppression of the host immune system to avoid side effects caused by immunosuppressive drugs. The permeability of alginate microcapsules is considered a suitable property for cell encapsulation, which allows diffusion of nutrients, waste, and therapeutic factors into and out of cells while masking them from the host immune response^8,9,10.

In the eye, encapsulated cells have been used in clinical trials for the constant delivery of biologics (i.e., growth factors^11,12 and growth factor antagonists¹³) for the treatment of retinitis pigmentosa or age-related macular degeneration. Other targets such as complement inhibitors¹⁴ are also currently being explored in preclinical settings.

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 in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).
3. Incubate the cells at 37 °C and 5% CO₂.
4. Replace the medium every 2–3 days.
5. Passage the cells after reaching 70%–80% confluence using standard tissue culture procedures.

2. Cell Encapsulation

1. Mix sodium alginate with deionized (DI) water for a final concentration of 2% w/v and purify by filtration with a 0.2 µm sterile syringe filter.
2. Prepare the cells to be mixed with the alginate solution by trypsinizing, centrifuging, and washing them with 10 mM HEPES buffered saline solution (pH = 7.4). Using a hemocytometer, count the cells and adjust the final cell concentration to 1 x 10⁶ in alginate solution.
   NOTE: The encapsulation process should be run inside a sterilized hood.
3. Load ~300 µL aliquots of alginate and cells mixture into a 3 mL syringe and attach it to a syringe pump. The solution will be pumped through a 30 G blunt tip needle to a sterile gelling bath placed in a sterile 50 mL beaker below the syringe tip at 7 mm for a needle to bath spraying distance.
4. The gelling bath contains a volume of 40 mL of 10 mM HEPES buffered saline containing 100 mM calcium chloride (CaCl₂) and 0.5% w/v poly-L-ornithine (PLO). The PLO is a secondary polymer coating that can be omitted or changed according to the needs of the investigator.
5. Adjust the voltage and flow rate and keep them both constant during the encapsulation process at 60 mm/h flow rate and 6.0 kV initial voltage to produce microcapsules size of ~150 µm.
6. Connect the clip of anode wire (red) of a high voltage generator needle tip to the needle and connect the ground clip (black) to the copper wire that is halfway submerged in the gelling bath. One batch of the alginate + cell mixture (1 mL) takes approximately 30 min to prepare the encapsulated cells (approximately 25,000 microcapsules).
7. Wash the formed microcapsules containing cells with washing solution (10 mM HEPES buffered saline containing 1.5 mM CaCl₂, pH = 7.4) twice. Do not use PBS for washing.
8. Incubate the encapsulated cells with 10% FBS supplemented DMEM media in a humidified incubator at 37 °C and 5% CO₂.
   NOTE: Encapsulation process instruments are depicted in Figure 1.

3. Confirmation that Encapsulation Does Not Affect Cell Viability

1. After incubating the encapsulated cells for 24 h in media, prepare a small sample of 500 µL (~30 microcapsules) for staining.
2. Wash the microcapsules 2x using washing solution (10 mM HEPES buffered saline containing 1.5 mM CaCl₂) and stain them for live-dead viability using a live/dead assay kit.
3. Prepare a staining mixture of calcein AM (acetoxymethyl) and ethidium homodimer-1 at final concentrations of 2 µM and 4 µM, respectively.
4. Add 2 mL of the staining mixture to the encapsulated cells and incubate for 30–45 min in the dark at room temperature (RT).
5. With careful aspiration, aspirate the staining solution and wash the microcapsules 2x with the washing solution. Use a fluorescent microscope system to observe and image the encapsulated cells.
   NOTE: Documentation of encapsulation is depicted in Figure 2.

4. Confirmation That Capsules Are the Appropriate Size for Delivery and Delivery of the Biologic

1. Intravitreal injections in a mouse eye are typically performed with a 27 G blunt-tip needle (inner diameter of 210 µm) attached to a 2.5 µL Hamilton syringe.
2. Generate capsules ranging from 100 to 200 µm, dilute them in serum-free media, and carefully pull them up into the Hamilton syringe. PBS is not a suitable vehicle, as alginate capsules will dissolve in PBS.
3. Slowly eject 1 µL drops containing capsules onto a microscope slide and determine their integrity using an upright bright-field microscope.
4. Adjust the capsule size (see step 2.3) by adjusting voltage and flow rate accordingly. Smaller microcapsules are produced in a nonlinear fashion by increasing voltage and slightly decreasing flow rate⁸. In our hands, capsules of 150 µm in diameter proved to be most suitable. Adjustment of capsule size is also dependent on keeping the alginate concentration constant while changing the other parameters.
5. Maintain a separate set of cells in capsules of the appropriate size in serum-free medium to determine the amount of secretion of the desired biologic. Use sensitive ELISAs or western blotting to determine the concentration of the biologics in the supernatant.
6. Determine the required amount of capsules that need to be injected based on the known PK/PD (pharmacokinetics/pharmacodynamics) of the therapeutic. In our hands, 10 capsules per mouse eye proved to be most efficacious.

5. Capsule Delivery into Mouse Vitreous

1. Perform intravitreal injections using a dissecting microscope. See Figure 3 for surgical set-up and materials used during the procedure. A detailed protocol for intravitreous injections can be found elsewhere¹⁶.
2. Anesthetize mice by intraperitoneal injection of xylazine and ketamine (20 mg/kg and 80 mg/kg) or other preferred anesthetics approved by the specific institution’s Animal Care and Use Committee. Ensure the appropriate depth of anesthesia using a toe pinch¹⁷.
3. Dilate the mouse pupils with phenylephrine HCL (2.5%) and atropine sulfate (1%) to allow for good visibility of the vitreous chamber and apply a lubricant eye gel to the eyes to keep them hydrated during the procedure.
4. Puncture the sclera at the limbus with a 26 G needle as the guide hole, making sure that 1) the needle is at a 45° angle with the eye and table and 2) the beveled tip is pointed upwards to avoid puncturing the lens.
5. Carefully inject the capsules using a 27 G blunt-tip needle attached to a Hamilton syringe at a 45° angle under visual inspection, making sure to avoid touching the lens with the needle. Injury of the lens will lead to cataract formation. The capsules should be visible in the vitreous using the dissecting microscope (Figure 4A).
6. After retraction of the needle, treat the injection site with antibiotic ointments neomycin and polymyxin B sulfates in addition to dexamethasone ophthalmic antibiotic ointment.
7. Apply goniotaire hypromellose demulcent ophthalmic solution (2.5%) to both eyes to prevent the corneas from drying out during the recovery period.
8. Place the mouse on a heating pad held at 37°C and monitor until fully awake.
9. A successful injection of encapsulated ARPE-19 cells should reveal the presence of intact capsules in the vitreous chamber of the mouse with only minor amounts of debris when imaging the eye by optical coherence tomography or other methods (Figure 4B).
10. The injected mouse eye, after a few days of recovery due to the surgery, is now ready for the experimental paradigm at hand.
    NOTE: The surgical set-up and documentation of capsules in the eye are depicted in Figure 3 and Figure 4, respectively.

Results

ARPE-19 cells are a spontaneously immortalized human RPE cell line that has been shown to be amenable to encapsulation and long-term survival upon implantation of capsules into the eye. The tools for alginate encapsulation are shown in Figure 1. In this study, it was demonstrated that upon encapsulation in alginate, the cells in alginate capsules were confirmed by bright-field imaging (Figure 2A). Live-dead assays were performed on the cells inside the capsules,...

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

List of materials used in this article

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 G, Small Hub RN NDL, custum length (12 mm), 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, 1 M	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 μm	VWR	28143-312
Syringe pump	GRASEBY	MS16A