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
2. Cell Encapsulation
3. Confirmation that Encapsulation Does Not Affect Cell Viability
4. Confirmation That Capsules Are the Appropriate Size for Delivery and Delivery of the Biologic
5. Capsule Delivery into Mouse Vitreous
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, demonstrating 90% viability post-encapsulation (Figure 2B). To ensure long-term viability of the cells inside capsules, capsules were dissolved in sodium citrate and cells were then re-plated (Figure 2C). After confirming in independent experiments that the stable transfected ARPE-19 cells expressed and secreted the biologic that was desired14, and after establishing the parameters for safely encapsulating cells, capsules were produced for intraocular injection.
Intravitreal injections into the mouse eye requires the use of a 27 G blunt-tip needle (inner diameter of 210 µm) and accurate delivery system to consistently inject the required small volume of 1 µL. The size of capsules that were not damaged during ejection through the 27 G needle was confirmed by microscopy (Figure 2A). An optimal size of 150 µm was identified. Intravitreal injection was performed under visual inspection, which allowed the visualization of capsules in the vitreous (Figure 4A). Likewise, OCT imaging was performed, which confirmed that a majority of capsules in the vitreous chamber of the mouse were intact with relatively minor amounts of debris (Figure 4B). In follow-up published experiments, it was confirmed that the desired biologic was present in the eye at therapeutically relevant doses, inhibiting the disease process under investigation14. These results confirmed that encapsulated cells can safely be injected into mouse eyes for the long-term delivery of biologics.
Figure 1: Encapsulation process instruments. The set-up includes (A) a 3 mL syringe, (B) 0.2 µm filters, (C) a 30 G, ½ inch blunt-tip needle, (D) an electronic syringe driver, (E) a high voltage generator, (F) the system set-up, and (G) a 7 mm fixed distance between the needle tip and gelling solution surface. Please click here to view a larger version of this figure.
Figure 2: Cell encapsulation. (A) Microcapsules filled with ARPE 19 cells. (B) Live/ dead assay: [a] red fluorescent color indicates dead cells, and [b] green fluorescent color indicates live cells. (C) Cultured cells after recovery from microcapsules beginning with a small cluster growing to confluency. Scale bars = 100 µm. Please click here to view a larger version of this figure.
Figure 3: Surgical set-up. A dissecting microscope (1) with a video camera (2) is used to visualize the procedure. The mouse is weighed (3) to administer the appropriate amount of anesthetic and placed on a small platform (4) to ease handling and injection. Eyes are dilated with mydriatics atropine and phenylepinephrin (5) and are kept hydrated with lubricant (6). A guide hole is punctured just outside the limbus using a 26 G needle (7). The glass syringe (8), loaded with appropriately diluted capsules (9), is placed at a 45° angle to the mouse eye and advanced through the guide hole, using a micromanipulator (10). The needle track as well as the capsules being released can be visualized [(11); see Figure 4]. Upon retraction of the needle, the cornea is moistened with hypromellose (12) and antibiotic ointment (13) applied to the injection site to avoid infection. Following the procedure, the mouse will be placed on a 37 °C heating pad and monitored until fully awake. Please click here to view a larger version of this figure.
Figure 4: Encapsulated cell technology to deliver ARPE-19 into the eye. (A) Injections are performed using a blunt 27 G needle attached to a Hamilton syringe. The needle track can be followed as the tip of the needle enters the eye, avoiding the lens and the capsules (arrowhead) can been visualized, magnified by the optical system of the mouse eye. It should be noted that the circular reflection of the light source. (B) Capsules (arrowhead) can be imaged in the vitreous using optical coherence tomography. Scale bars = 200 µm. Panel B was reprinted with permission from Annamalai et al.14 Please click here to view a larger version of this figure.
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 |
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.
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.
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.