Current in vitro models for evaluating contact lenses (CLs) and other eye-related applications are severely limited. The presented ocular platform simulates physiological tear flow, tear volume, air exposure and mechanical wear. This system is highly versatile and can be applied to various in vitro analyses with CLs.
Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic physiological tear volumes.7 The traditional model also lacks the natural tear flow component and the blinking reflex, both of which are defining factors of the ocular environment. The development of a novel model is described in this study, which consists of a unique 2-piece design, eyeball and eyelid piece, capable of mimicking physiological tear volume. The models are created from 3-D printed molds (Polytetrafluoroethylene or Teflon molds), which can be used to generate eye models from various polymers, such as polydimethylsiloxane (PDMS) and agar. Further modifications to the eye pieces, such as the integration of an explanted human or animal cornea or human corneal construct, will permit for more complex in vitro ocular studies. A commercial microfluidic syringe pump is integrated with the platform to emulate physiological tear secretion. Air exposure and mechanical wear are achieved using two mechanical actuators, of which one moves the eyelid piece laterally, and the other moves the eyeballeyepiece circularly. The model has been used to evaluate CLs for drug delivery and deposition of tear components on CLs.
Two significant areas of interest within the contact lens (CL) arena include discomfort and the development of novel CL applications. Elucidating the mechanisms underlying CL discomfort is an issue that has eluded the field for decades.8 The development of novel, functional CLs, such as drug-delivery devices1,3,9 and biosensors,10-12 is an area of growing interest, with substantial potential markets. In both circumstances, a sophisticated in vitro model would provide relevant information to assist with selecting appropriate lens materials or design characteristics during the development phase. Unfortunately, current in vitro models for evaluating CLs and other eye related applications are relatively crude and unsophisticated. Traditionally, in vitro CL studies evaluating tear film deposition or drug delivery are performed in static, large volume vials containing a fixed fluid volume, which greatly exceeds physiological amounts. Furthermore, this simple model lacks the natural tear flow component and the blinking reflex, both of which are defining factors of the ocular environment.
The development of a sophisticated, physiologically relevant eye "model" will necessitate a multi-disciplinary approach and require substantial in vivo validation. For these reasons, the fundamental framework for our in vitro eye model is highly versatile, such that the model can be continually improved through future upgrades and modulations. To date, the model is capable of simulating tear volume, tear flow, mechanical wear and air exposure. The aim is to create an in vitro model that will provide meaningful results, which is predictive and complimentary to in vivo and ex vivo observations.
All experiments were completed in accordance and compliance with all relevant guidelines outlined by the University of Waterloo's animal research ethics committee. The bovine eyes are generously donated from a local abattoir.
1. Eye Model
2. Blink-platform
The synthesized eye molds obtained from the machine shop and from 3-D printing are shown in Figure 1. These molds can be used with a variety of polymers, such as PDMS and agarose, to produce eyepieces with the desired properties. The motioned assembly of the eye model platform with a microfluidic syringe pump is shown in Figure 2. The platform simulates mechanical wear via the rotation of the eyeball piece, and air exposure through the lateral in and out motion of the eyelid piece. Tear fluid is infused into the eyelid from a microfluidic pump at the desired flow rate, and the flow-through fluid can be collected in a 12-well plate.
The procedure for dissection of a bovine lens, and mounting onto a PDMS eyepiece is depicted in Figure 3. The excess tissues are separated from the eye and discarded, followed by the removal of the conjunctiva. The removal of the cornea begins with an incision into the sclera near the limbus. Figure 4 shows the variety of eyepieces that could be used for various in vitro analyses. The mounted eyeball pieces shown are synthesized from PDMS, agar, and an ex-vivo bovine cornea mounted on a PDMS eyeball piece.
Figure 5 depicts a study evaluating the release of an antibiotic, moxifloxacin, from CLs.18 When measured in the traditional vial model, drug release occurs within the first 2 hr followed by a plateau phase. In contrast, the novel eye model shows drug release to be slow and sustainable for up to 24 hr.18 A study evaluating the deposition of cholesterol on CLs is shown in Figure 6. The cholesterol in the study was fluorescently tagged in the form of NBD-cholesterol (7-nitrobenz-2-oxa-1,3-diazol-4-yl-cholesterol), and deposition was imaged using laser scanning confocal microscopy. The results indicate that there are substantial differences when the deposition studies are performed in a vial as compared to the eye model.
Figure 1. Eyepiece molds. (A) Eyeball piece mold from machine shop. (B) Eye lid mold from 3-D printing. Please click here to view a larger version of this figure.
Figure 2. An in vitro ocular platform. (A) Circular motion simulates mechanical wear. (B) Lateral motion produces intermittent air exposure. (C) Tear fluid infusion into eyelid. (D) Collecting well plate. Please click here to view a larger version of this figure.
Figure 3. Dissection and incorporation of bovine cornea. (A) Removal of excess tissue. (B) Removal of conjunctiva. (C) Incision into the limbus region. (D) The excised cornea can be stored or mounted on a PDMS eye ball piece. Please click here to view a larger version of this figure.
Figure 4. Sample eyepieces. Sample of PDMS eye piece with a contact lens, an agar eye piece, and ex vivo bovine cornea mounted eye piece. Please click here to view a larger version of this figure.
Figure 5. Drug delivery using the in vitro ocular platform. Release of moxifloxacin from daily disposable contact lenses from (A) a large volume static vial and (B) the eye model (Re-print with permission from the Association for Research in Vision and Ophthalmology).18 All data are reported as mean ± standard deviation. Please click here to view a larger version of this figure.
Figure 6. Cholesterol deposition using the in vitro ocular platform. Confocal images showing a cross-section of etafilcon A, nelfilcon A, nesofilcon A, ocufilcon B, delefilcon A, somofilcon A, narafilcon A after 4 hr incubation with NBD-cholesterol in the vial and eye model. Please click here to view a larger version of this figure.
There are three critical steps within the protocol that require special attention: design and production of molds (section 1.1), platform assembly (section 2.2.1-2.2.3), and monitoring the experimental run (section 2.2.4-2.2.7). In terms of the design and production of molds (section 1.1), the eyeball piece should be designed according to the dimensions of a human cornea. However, it may require multiple prototypes of the mold before an eyeball piece can be created that perfectly fits a commercial contact lens (CL). In addition, the 250 µm needs to be maintained when the eyeball and eyelid piece are in contact to ensure the tear fluid flows smoothly throughout the entire eye model when a CL is present. This distance could be changed in future iterations, but should not be less than 150 µm to allow for enough spacing to fit a CL. The platform assembly (section 2.2.1-2.2.3) requires careful attention such that the eyeball and eyelid piece come into contact during the blink motion. If the eyepieces are not in perfect contact, then simulation of a closed eyelid and mechanical rubbing fails. The operator should observe the platform in motion for a few cycles to ensure that both the eyeball and eyelid are in contact, and that rubbing occurs as programmed. The current platform is designed to run continuously over one month, but an operator should always check on the stability of the system every 24 hr when running an experiment (section 2.2.4-2.2.7). This is important as the current platform does not possess a temperature or humidity control, and fluctuations in these parameters could dry up the CLs. If this occurs, place the eye model within a controlled humidity and temperature chamber. In addition, for drug delivery experiments, the collected flow-through fluid should be analyzed or stored at least every 2 hr to avoid significant evaporation of the sample.
There are currently two limitations of the presented eye model. The first limitation is in regards to exposure to the surrounding environment. Currently, because the eye pieces are not enclosed in a controlled chamber, changes such as temperature and humidity in the work area will influence various aspects of the experiments. For instance, if the environment is too dry, then the CLs dry up quicker and could separate from the eyeball piece, or the flow-through fluid could evaporate. To address this problem, future iterations will house the eye model in a controlled temperature and humidity chamber. The second limitation pertains to the complexity eyeball piece. Currently, the eyepieces are simple, consisting of either PDMS or agarose, neither of which truly represents corneal surface properties. Future work will aim to produce eye models which closer mimics the corneal surface structures.
In vitro ocular research is generally viewed as the preceding testing phase to in vivo research. However, it is important to keep in mind that in vitro research can also be complementary to in vivo data, providing critical insights that otherwise cannot be achieved from in vivo studies alone. Regrettably, the current in vitro models for testing CLs are rudimentary and lack several key components to adequately mimic the in vivo environment. For instance, in vitro CL studies are performed in vials containing 2-5 ml of phosphate buffered saline,1-6 which greatly exceeds physiological tear volumes at 7.0 ± 2 µl.7 Moreover, two important factors of the ocular environment, natural tear flow and the blinking reflex, are absent from the simple static vial model. The limitations of the conventional vial model have been recognized by researchers, and attempts have been made to create unique in vitro eye models simulating the ocular environment, by including a microfluidic tear replenishment component20-24 and/or intermittent air exposure.25,26 Not surprisingly, the results generated from these experiments are very different than those obtained with the conventional vial model, and may more closely resemble in vivo data.20-25 Thus, developing an intricate in vitro eye model to examine CLs will provide new insights on the interaction of lens materials with the ocular surface, and help facilitate the development of new materials and new applications for CLs in the coming decades.
Arguably, one of the most debated aspects of the in vitro eye model is whether the eye resembles an infinite sink, which is particularly important when it comes to drug delivery from CLs. Under infinite sink conditions, the volume of the surrounding solution is significantly higher than the drug saturation volume, such that drug release is not affected by the drug's solubility.27 Advocates for the vial as an acceptable eye model argue that the cornea, conjunctiva, and surrounding ocular tissues together function as an infinite sink. While in theory this may be true, the drug must first dissolve into the tear fluid. This rate limiting step is likely not a sink condition, and will be dependent on both tear volume and flow as simulated by our model.
The unique identity of the presented model lies in its ability to emulate the tear film. By adopting a two-piece design, a "corneal/scleral" eyeball section and an "eyelid", it is possible to create an evenly spread thin layer of tear film across the eyeball piece when both pieces come into contact. To further simulate the ocular surface, mechanical wear and air exposure is incorporated into the model through two mechanical actuators. As the eyelid piece moves laterally, it simulates the closing of the eye and intermittent air exposure. The rotation of the eyeball simulates the mechanical wear produced during blinking. The system is coupled with a microfluidic pump, which infuses the eye model with tear fluid at a physiological flow rate or any other desired flow rate. The tear film is formed each time the two pieces come into contact, and tear break-up occurs when the two pieces separate.
The aim is to create a universal testing platform to evaluate CLs for various in vitro analyses. In order to be versatile, the eyeball pieces can be synthesized from various polymers, such as polydimethylsiloxane (PDMS) or agar. For simple ocular studies, these polymers, which represent hydrophobic and hydrophilic surfaces respectively, will suffice. However, as more complex analyses are required, for example ocular drug penetration or toxicity studies, the eye pieces will need to be further modified. These additional modifications to the model, such as the inclusion of an ex vivo cornea as shown, are relatively feasible. However, further validation studies are required, and future work will aim to improve the validity of this model by comparing it with in vivo models.
The authors have nothing to disclose.
The authors would like to acknowledge our funding source NSERC 20/20 Network for the Development of Advanced Ophthalmic Materials.
Arduino Uno R3 (Atmega328 – assembled) | Adafruit | 50 | Board |
Stepper motor | Adafruit | 324 | Motor and Motor shield |
Equal Leg Coupler 1.6mm 1/16" | VWR | CA11009-280 | 50 pcs of tube connector |
Tubing PT/SIL 1/16"x1/8" | VWR | 16211-316 | Case of 50feet |
PDMS | Dow Corning | Sylgard 184 Solar Cell Encapsulation | |
Agarose, Type 1-A, low EEO | Sigma-Aldrich | A0169-25G | |
PHD UltraTM | Harvard Apparatus | 703006 | MicroFluidic Pump |
Bovine cornea | Cargill, Guelph/ON | ||
Soldidworks | Dassault Systemes | Software | |
3-D printing | University of Waterloo – 3D Print Centre | ||
Dissection tools | Fine Science Tools | General dissection tools | |
Medium 199 | Sigma-Aldrich | Culture medium storage for cornea | |
Fetal bovine serum | Thermo Fisher | Add to culture medium, 3% total volume |