The overall goal of this in vitro eye model is to simulate physiological tear volume, natural tear flow, air exposure, and mechanical wear produced during blinking, to assess the performance of contact lenses. This method can help answer key questions in the contact lens field, such as predicting the performance of contact lenses, and related ophthalmic devices on the eye. The main advantage of this technique is that it provides a simple and cost effective in vitro model to simulate multiple on-eye factors, which is currently not available.
Though this method is designed to provide insight into contact lens performance, it can also be used to test other ophthalmic systems, such as the glucose smart contact lens currently being developed by Google and Novartis. Visual demonstration of this method is important to understand how the eye model operates and how it can be used effectively in various research scenarios. The eyelid and eyeball parts of the model are made from PDMS.
First, prepare the PDMS. Using a syringe, transfer 10 milliliters of PDMS base into a 15 or 50 milliliter centrifuge tube. Then weigh out and mix in a 10%weight by volume addition of elastomer solution.
Use a stir rod to mix the solution well. Now, pour the PDMS solution into the eyeball and eyelid molds. Fill the molds carefully to avoid trapping air bubbles.
Allow the PDMS to settle at room temperature overnight, during which it will polymerize and small air bubbles will dissolve out of the mold. The next day, transfer the molds to an oven set to 75 degrees Celsius for an hour. Then, put the molds in a negative 80 degrees Celsius freezer for a few minutes.
The PDMS will shrink a little. Then, release the samples from the mold using a spatula. Next, connect a PTFE tube to an equal leg coupler, and attach it to the eyelid piece.
This tube introduces fluids between the samples. Now synthesize the eyeball piece from agarose or a similar agar. First, make 100 milliliters of 2%agarose in solution.
When the solution cools to about 40 degrees Celsius, pour it into the eyeball mold. Excess agar can be frozen for later use. Allow the solution to cool for 30 minutes at room temperature, and then remove the eyeball pieces with a spatula.
For microbiology studies, sterilize the eyeball molds with an autoclave or UV irradiation. After running the ventilation in the hood for at least 10 minutes, sanitize the surfaces with 70%ethanol alcohol. Perform this procedure with the bovine corneas under sterile conditions in a laminar flow hood.
Dissect the eyes immediately after they have been harvested. Use only autoclaved tools, and place nothing within four inches of the front edge of the workspace. Begin working with the bovine eye by immersing it in 0.5%povidone iodine solution for two minutes.
Then remove the eye from the povidone iodine solution, and rinse the eye with PBS at a pH of 7.4. Now use forceps to gently transfer the eye to a glass petri dish, corneal face up. Remove the excess muscle and fatty tissue by cutting at the scleral attachment points with blunt-end dissection scissors.
Dispose of the excess tissue into a sterile beaker designated for animal waste. Next, using microscissors, remove the conjunctiva from the eye. Wrap the eye with sterile gauze, but keep the gauze at least a centimeter from the limbus.
Correct execution of this step is critical for success. Then using a scalpel, incise the sclera about two millimeters from the limbus region to avoid penetrating the underlying choroid and vitreous body. Next, carefully extend the incision 360 degrees around, without deforming the cornea from its natural curvature.
Now, using fine forceps, remove the cornea from the eye. Take extra care when performing this step. Then, carefully remove any adhering uveal tissue, if there is any remaining, from the cornea, and rinse the cornea with PBS.
Store the cornea at 31 degrees Celsius in culture medium, such as medium 199 containing 3%fetal bovine serum. To add the cornea to the experimental model, rest it on the eyeball piece and clamp the two pieces together. Details on the design and production of the eyelid piece for the model are covered in the text protocol.
This section covers its assembly, and the addition of artificial tear solution. To begin, secure the synthesized eyeball and eyelid pieces to the eye model. Next, attach tubing connected to a syringe and a microfluidic pump to the model, and check if consistent movement is generated.
First, prime the tubing with artificial tear solution. Then, manually put the eyeball and the eyelid in contact on a level plane. Now, start the pump at a physiological flow between one and 1.5 microliters per minute.
For drug delivery experiments, place the drug-containing contact lens on the eyeball piece. Allow the flow-through fluid to drip into a 12-well plate. At the desired time intervals, quantify the analyte or drug concentration in the flow-through using standard methods.
To evaluate the deposition of tear components on a contact lens, run the experiment for the desired time, discarding the flow-through, remove the contact lens, and analyze it, as needed. The described model is compatible with a variety of eye pieces that could be used for various in vitro analyses. In this example, the mounted eyeball pieces are synthesized from PDMS and agar, and the cornea used is an ex vivo bovine cornea.
The release of an antibiotic moxifloxacin from contact lenses was evaluated. When measured in the traditional vial model, drug release occurred within two hours, followed by a plateau phase. In contrast, the novel eye model showed that drug release was slow and sustainable for up to 24 hours.
The model was next used to study the deposition of cholesterol on contact lenses. The cholesterol in the study was fluorescently tagged and viewed using laser scanning confocal microscopy. Substantial differences were seen in deposition studies performed in a vial compared to the eye model.
Once mastered, this eye model would take 10 minutes to set up if it is used properly. While attempting this procedure, it is important to remember to make sure that the tear fluid drops down into the collecting well plate. Following this procedure, other methods like confocal microscopy or spectophotometry can be performed to evaluate the deposition of tear components on the material or drug illusion from the lens.
After its development, this technique paved the way for researchers in the field of contact lenses to explore tear fluid deposition and drug delivery. Don't forget that when working with biologicals or chemicals to be safe and sterile.
