Development of an In Vitro Ocular Platform to Test Contact Lenses

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 devices^1,3,9 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.

Protokół

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

1. Design and Production of Molds¹³
   1. Design the eye models according to the average physiological dimensions of human adult eyes.¹³
   2. Leave a gap of 250 µm between the eyeball and the eyelid pieces of the eye model. Design the respective molds using computer-aided design (CAD) software.
   3. Create new .cad file or .sldprt file with AutoCAD or Solidworks. Create 3D models of the human eyeball/eyelid. Create molds of the models and save the molds as .stl files.
   4. Import .stl files into 3D printer software (e.g., makeware for replicator2). Specify parameters of the print (location, sparseness, scale, orientation, smoothness, etc.)¹³.
   5. Save the file as G-code file for 3D printers to read. Select materials such as PLA (polylactic acid), ABS (acrylonitrile butadiene styrene), PC (polycarbonate), or a combination thereof, to print the molds¹³.
   6. Install desired filament of the material of choice. Import the G-code file into the 3D printer to read. Print the mold.
      NOTE: Alternatively, produce the eye molds using a computer numerical controlled (CNC) machine, if a smoother surface on the eye model is desired. For CNC mold production, materials for molds are no longer limited to thermal plastics, but extend to metal, ceramics, and chemically resistive polymers such as Polytetrafluoroethylene.
   7. Open the CNC software interface that is connected to a cutting drill. Construct 3D molds according to front, top, side, and perspective views of the previously-constructed eyeball/eyelid model molds in control software interface. Select appropriate parameters for the machining (bit size, substrate material, material thickness) and proceed to cut the mold.
2. Synthesis of Eyepieces Using PDMS
   1. Using a syringe, measure 10 ml volume of PDMS (polydimethylsiloxane) base and fill it into a 15-50 ml centrifuge tube. Add 10% w/v of the elastomer solution by total weight of PDMS. Using a stirring rod, mix the solutions well.
   2. Pour the PDMS solution into the eyeball and eyelid molds. Allow the PDMS to settle at RT O/N (or for at least 12 hr) to start the polymerization and to allow bubbles to dissolve out of the polymer.
      NOTE: Ensure that there are no bubbles left in the PDMS that might rise or expand.
   3. Subsequently, put the molds into a 75 °C (167 °F) oven for 1 hr, or 150 °C (302 °F) for 5 min. For a softer gel, let the PDMS sit at RT for at least 48 hr to completely polymerize.
   4. Put the samples in a freezer for a few min; this will shrink the PDMS and simplify the removal of the samples from the molds. Extract the eyepieces from the molds using a thin spatula.
   5. For the delivery of solution into the space between the eyeball and eyelid pieces, connect a 1/16" x 1/8" polytetrafluoroethylene tube with a 1/16" equal leg coupler tube connector and attach it to the eyelid piece at the tubing hole.
3. Synthesis of Eyeball Piece Using Agarose
   NOTE: The eyeball piece can be synthesized using other polymers such as agarose. The following procedure can also be modified to produce eye pieces from a variety of agar types, such as PDA (potato dextrose agar) or SDA (sabouraud dextrose agar).
   1. To produce a 2% (2 g/100 ml) gel, measure 2 g of agarose and mix with 100 ml of ultrapure water. Bring the solution to a boil (100 ºC) such that the agarose dissolves completely. Let the solution cool down for 5 min.
   2. Pour the solution into the eyeball mold and allow the solution to cool for 30 min at RT. Remove the eyeball pieces with a spatula. Store the eyeball agar in a -20 °C freezer for later use. For microbiology studies, sterilize the eyeball molds by autoclaving and/or UV-irradiation.
4. Incorporation of Bovine Cornea on PDMS Eyeball
   NOTE: This protocol has been adapted from Parekh et al.¹⁴
   1. Perform the dissection and incorporation of the bovine corneas in sterile conditions under a laminar flow hood. Acquire the eyes and dissect them on the same day.
   2. Turn the flow hood on for 10 min prior to use and sanitize with 70% ethanol alcohol. Ensure that all materials and instruments are sterile by autoclaving at 273 °F/133 °C for 45 min, and positioned no less than 4 inches from the flow hood entrance.
   3. Immerse the bovine eye in a beaker containing a diluted povidone-iodine solution for 2 min. Rinse the eye in a beaker containing phosphate buffered saline (PBS) pH 7.4. Using forceps gently place the eye on a glass petri dish, corneal face up.
   4. 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.
   5. Using micro-scissors, remove the conjunctiva from the eye. Wrap the eye with sterile gauze, maintaining a distance of at least 1 cm from the limbus.
   6. Using a scalpel, incise the sclera approximately 2 mm from the limbus region and superficially so as to avoid penetration of the underlying choroid and vitreous body. Carefully extend the incision by 360° using a scalpel or dissection scissors without deforming the cornea from its natural curvature.
   7. With fine forceps, remove the cornea from the eye. Using forceps, carefully remove any adhering uveal tissue and rinse cornea with PBS.
   8. Store the cornea at 31ºC in a sterile container with culture medium (such as Medium 199) containing 3% Fetal Bovine Serum to maintain tissue moisture and cell nourishment.
   9. Prior to experimentation, rest the excised cornea on the PDMS eyeball, and clamp the two pieces together with a specialized clip-on.

2. Blink-platform

1. Design and Production of the Blink-platform
   NOTE: The blink-platform is composed of three functional parts: eye model (described in section 1), gear system, and electronic system.
   1. Design and manufacture the blink platform using CAD and 3D printing, similar to that described for the eye model (section 1.1). Design the gear system such that it translates simple rotation of motors into the lateral and rotational motions of the eyepieces.¹⁵
   2. Using the pinion and gear mechanism, translate rotational motion of a stepper motor into the lateral motion of a pinion, which is connected to the eyelid pieces.
   3. Using the conjugate gear system, amplify one rotational motion from a stepper motor into three (or more) rotational motions for three different eyeball pieces.
   4. Align the two gear systems, one for the eyelid and one for eyeball, so that the distance between the two are constant. Assemble the electronic system with a microcontroller, motor shield, and two motors.
      NOTE: Use two stepper motors to provide rotational motors, which is translated by the gear system into a blinking motion.
   5. Connect the two stepper motors with a system consisting of a motor shield stacked on the microcontroller. Connect and configure the electronic components to work with open source software products.
   6. Program the system to control motor parameters such as rounds per minute (RPM), number of rounds forward, number of rounds backward, and turning style.
      NOTE: Refer to the supplementary "Arduino code file" for details.
   7. Download the system software from the manufacturers' website.
   8. Install the software and open it. Write the code to control stepper motors in the desired configuration. Connect the system with a source to power the electronic system so that the motors move in the desired manner as defined by the researcher.
      NOTE: Refer to the supplementary "Arduino code file".
2. Assembly with Microfluidics (Artificial Tear Solution)
   1. Take the synthesized eyeball and eyelid pieces and slip them onto their corresponding clip-ons for the eye-model. Connect the tubing that is joined with a syringe and positioned on the microfluidic pump with the eyelid piece (section 1.2.5). Test run the platform and check for consistent movement.
   2. Prime the tubing and check for a steady flow of artificial tear solution (ATS). The recipe for ATS has been previously reported.¹⁶
   3. Manually move the eye-model parts together on a level plane, such that the eyeball and the eyelid are in contact. Set the flow rate of the microfluidic pump to desired values. Set physiological flow rates to 1-1.5 µl/min.¹⁷
   4. Start the pump and the actuators to begin experiment. For drug delivery experiments, place the drug-containing contact lens on the eyeball piece.
   5. Allow the flow-through fluid to drip into a 12-well plate. At the desired set time intervals, quantify the analyte or drug concentration using common detection methods such as UV-Vis spectroscopy or fluorescence.^1,4,18
   6. For studies evaluating deposition of tear components on contact lenses, place the contact lens on the "eyeball" piece. Collect the flow-through fluid, which can be discarded.
   7. After the desired time intervals, remove the contact lens from the eyeball piece and prepare the lens for further analysis such as confocal microscopy.

Wyniki

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 ...

Dyskusje

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 a...

Materiały

Name	Company	Catalog Number	Comments
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