The authors would like to acknowledge our funding source NSERC 20/20 Network for the Development of Advanced Ophthalmic Materials.

All experiments were completed in accordance and compliance with all relevant guidelines outlined by the University of Waterloo&#39;s animal research ethics committee. The bovine eyes are generously donated from a local abattoir.
1. Eye Model
<ol>
	<li>Design and Production of Molds13
	<ol>
		<li>Design the eye models according to the average physiological dimensions of human adult eyes.13</li>
		<li>Leave a gap of 250 &#181;m between the eyeball and the eyelid pieces of the eye model. Design the respective molds using computer-aided design (CAD) software.</li>
		<li>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.</li>
		<li>Import .stl files into 3D printer software (e.g.,&#160;makeware for replicator2). Specify parameters of the print (location, sparseness, scale, orientation, smoothness, etc.)13.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Synthesis of Eyepieces Using PDMS
	<ol>
		<li>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.</li>
		<li>Pour the PDMS solution into the eyeball and eyelid molds. Allow the PDMS to settle at RT&#160;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.</li>
		<li>Subsequently, put the molds into a 75 &#176;C (167 &#176;F) oven for 1 hr, or 150 &#176;C (302 &#176;F) for 5 min. For a softer gel, let the PDMS sit at RT for at least 48 hr to completely polymerize.</li>
		<li>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.</li>
		<li>For the delivery of solution into the space between the eyeball and eyelid pieces, connect a 1/16&#34; x 1/8&#34; polytetrafluoroethylene tube with a 1/16&#34; equal leg coupler tube connector and attach it to the eyelid piece at the tubing hole.</li>
	</ol>
	</li>
	<li>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).
	<ol>
		<li>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 &#186;C) such that the agarose dissolves completely. Let the solution cool down for 5 min.</li>
		<li>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 &#176;C freezer for later use. For microbiology studies, sterilize the eyeball molds by autoclaving and/or UV-irradiation.</li>
	</ol>
	</li>
	<li>Incorporation of Bovine Cornea on PDMS Eyeball 
	NOTE: This protocol has been adapted from Parekh et al.14
	<ol>
		<li>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.</li>
		<li>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 &#176;F/133 &#176;C for 45 min, and positioned no less than 4 inches from the flow hood entrance.</li>
		<li>Immerse the bovine eye in a beaker containing a diluted povidone-iodine solution for 2&#160;min. Rinse the eye in a beaker containing phosphate buffered saline (PBS) pH 7.4.&#160;Using forceps gently place the eye on a glass petri dish, corneal face up.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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&#176; using a scalpel or dissection scissors without deforming the cornea from its natural curvature.</li>
		<li>With fine forceps, remove the cornea from the eye. Using forceps, carefully remove any adhering uveal tissue and rinse cornea with PBS.</li>
		<li>Store the cornea at 31&#186;C in a sterile container with culture medium (such as Medium 199) containing 3% Fetal Bovine Serum to maintain tissue moisture and cell nourishment.</li>
		<li>Prior to experimentation, rest the excised cornea on the PDMS eyeball, and clamp the two pieces together with a specialized clip-on.</li>
	</ol>
	</li>
</ol>
2. Blink-platform
<ol>
	<li>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.
	<ol>
		<li>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</li>
		<li>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.</li>
		<li>Using the conjugate gear system, amplify one rotational motion from a stepper motor into three (or more) rotational motions for three different eyeball pieces.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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 &#34;Arduino code file&#34; for details.</li>
		<li>Download the system software from the manufacturers&#39; website.</li>
		<li>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 &#34;Arduino code file&#34;.</li>
	</ol>
	</li>
	<li>Assembly with Microfluidics (Artificial Tear Solution)
	<ol>
		<li>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.</li>
		<li>Prime the tubing and check for a steady flow of artificial tear solution (ATS). The recipe for ATS has been previously reported.16</li>
		<li>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 &#181;l/min.17</li>
		<li>Start the pump and the actuators to begin experiment. For drug delivery experiments, place the drug-containing contact lens on the eyeball piece.</li>
		<li>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</li>
		<li>For studies evaluating deposition of tear components on contact lenses, place the contact lens on the &#34;eyeball&#34; piece. Collect the flow-through fluid, which can be discarded.</li>
		<li>After the desired time intervals, remove the contact lens from the eyeball piece and prepare the lens for further analysis such as confocal microscopy.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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 &#181;m 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 &#181;m 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 &#177; 2 &#181;l.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&#39;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 &#34;corneal/scleral&#34; eyeball section and an &#34;eyelid&#34;, 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.

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 &#34;model&#34; 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.

<table><tbody><tr><td>Arduino Uno R3 (Atmega328 - assembled)</td><td>Adafruit</td><td>50</td><td>Board</td></tr><tr><td>Stepper motor</td><td>Adafruit</td><td>324</td><td>Motor and Motor shield</td></tr><tr><td>Equal Leg Coupler 1.6mm 1/16&quot;</td><td>VWR</td><td>CA11009-280</td><td>50 pcs of tube connector</td></tr><tr><td>Tubing PT/SIL 1/16&quot;x1/8&quot;</td><td>VWR</td><td>16211-316</td><td>Case of 50feet</td></tr><tr><td>PDMS</td><td>Dow Corning</td><td>Sylgard 184 Solar Cell Encapsulation</td><td></td></tr><tr><td>Agarose, Type 1-A, low EEO</td><td>Sigma-Aldrich</td><td>A0169-25G</td><td></td></tr><tr><td>PHD UltraTM</td><td>Harvard Apparatus</td><td>703006</td><td>MicroFluidic Pump</td></tr><tr><td>Bovine cornea</td><td>Cargill, Guelph/ON</td><td></td><td></td></tr><tr><td>Soldidworks</td><td>Dassault Systemes</td><td></td><td>Software</td></tr><tr><td>3-D printing</td><td>University of Waterloo - 3D Print Centre</td><td></td><td></td></tr><tr><td>Dissection tools</td><td>Fine Science Tools</td><td></td><td>General dissection tools</td></tr><tr><td>Medium 199</td><td>Sigma-Aldrich</td><td></td><td>Culture medium storage for cornea</td></tr><tr><td>Fetal bovine serum</td><td>Thermo Fisher</td><td></td><td>Add to culture medium, 3% total volume</td></tr></tbody></table>

development of an in vitro ocular platform to test contact lenses

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.

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.
<img alt="Figure 1" src="/files/ftp_upload/53907/53907fig1.jpg" /> 
Figure 1. Eyepiece molds. (A) Eyeball piece mold from machine shop. (B) Eye lid mold from 3-D printing. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53907/53907fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53907/53907fig2.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53907/53907fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53907/53907fig3.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53907/53907fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53907/53907fig4.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53907/53907fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53907/53907fig5.jpg" /> 
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 &#177; standard deviation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53907/53907fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/53907/53907fig6.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53907/53907fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Development of an In Vitro Ocular Platform to Test Contact Lenses at JoVE.com

Development of an In Vitro Ocular Platform to Test Contact Lenses

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.

Development of an In Vitro Ocular Platform to Test Contact Lenses

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic ...

development-of-an-in-vitro-ocular-platform-to-test-contact-lenses

Nanyang Technological University

Research

JoVE Journal

Bioengineering

10.5K Views.  University of Waterloo.  Huidige in vitro modellen voor het beoordelen contactlenzen (CL's) en andere eye toepassingen ernstig beperkt. De gepresenteerde oculaire platform simuleert fysiologische traanvocht, scheur volume, belichting lucht en mechanische slijtage. Dit systeem is zeer veelzijdig en kan worden toegepast op diverse in vitro analyses CL's. 

Video: Development of an In Vitro Ocular Platform to Test Contact Lenses

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells&#8217; native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens1. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment.

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to ...

Developmental Biology

Synthesis of Soft Polysiloxane-urea Elastomers for Intraocular Lens Application

This study discusses a synthesis route for soft polysiloxane-based urea (PSU) elastomers for their applications as accommodating intraocular lenses (a-IOLs). Aminopropyl-terminated polydimethylsiloxanes (PDMS) were previously prepared via the ring-chain equilibration of the cyclic siloxane octamethylcyclotetrasiloxane (D4) and 1,3-bis(3-aminopropyl)-tetramethyldisiloxane (APTMDS). Phenyl groups were introduced into the siloxane backbone via the copolymerization of D4 and 2,4,6,8-tetramethyl-2,4,6,8-tetraphenyl-cyclotetrasiloxane (D4Me,Ph). These polydimethyl-methyl-phenyl-siloxane-block copolymers were synthesized for increasing the refractive indices of polysiloxanes. For applications as an a-IOL, the refractive index of the polysiloxanes must be equivalent to that of a young human eye lens. The polysiloxane molecular weight is controlled by the ratio of the cyclic siloxane to the endblocker APTMDS. The transparency of the PSU elastomers is examined by the transmittance measurement of films between 200 and 750 nm, using a UV-Vis spectrophotometer. Transmittance values at 750 nm (upper end of the visible spectrum) are plotted against the PDMS molecular weight, and &#62; 90% of the transmittance is observed until a molecular weight of 18,000 g&#183;mol&#8722;1. Mechanical properties of the PSU elastomers are investigated using stress-strain tests on die-cut dog-bone-shaped specimens. For evaluating mechanical stability, mechanical hysteresis is measured by repeatedly stretching (10x) the specimens to 5% and 100% elongation. Hysteresis considerably decreases with the increase in the PDMS molecular weight. In vitro cytotoxicity of some selected PSU elastomers is evaluated using an MTS cell viability assay. The methods described herein permit the synthesis of a soft, transparent, and noncytotoxic PSU elastomer with a refractive index approximately equal to that of a young human eye lens.

This study describes synthetic routes for aminopropyl-terminated polydimethylsiloxanes and polydimethyl-methyl-phenyl-siloxane-block copolymers and for soft polysiloxane-based urea (PSU) elastomers. It presents the application of PSUs as accommodating an intraocular lens. An evaluation method for in vitro cytotoxicity is also described.

This study discusses a synthesis route for soft polysiloxane-based urea (PSU) elastomers for their applications as accommodating intraocular lenses ...

Chemistry

Anterior Segment Organ Culture Platform for Tracking Open Globe Injuries and Therapeutic Performance

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and medical intervention in rural environments and military medicine applications where ophthalmic care is not readily available. Untreated injuries are susceptible to infection after the eye has lost its watertight seal, as well as loss of tissue viability due to intraocular hypotension. Therapeutics to temporarily seal open globe injuries, if properly developed, may be able to restore intraocular pressure and prevent infection until proper ophthalmic care is possible. To facilitate product development, detailed here is the use of an anterior segment organ culture open globe injury platform for tracking therapeutic performance for at least 72 h post-injury. Porcine anterior segment tissue can be maintained in custom-designed organ culture dishes and held at physiological intraocular pressure. Puncture injuries can be created with a pneumatic-powered system capable of generating injury sizes up to 4.5 mm in diameter, similar to military-relevant injury sizes. Loss of intraocular pressure can be observed for 72 h post-injury confirming proper injury induction and loss of the eye&#39;s watertight seal. Therapeutic performance can be tracked by application to the eye after injury induction and then tracking intraocular pressure for multiple days. Further, the anterior segment injury model is applicable to widely used methods for functionally and biologically tracking anterior segment physiology, such as assessing transparency, ocular mechanics, corneal epithelium health, and tissue viability. Overall, the method described here is a necessary next step toward developing biomaterial therapeutics for temporarily sealing open globe injuries when ophthalmic care is not readily available.

Open globe eye injuries may go untreated for multiple days in rural or military-relevant scenarios, resulting in blindness. Therapeutics are needed to minimize loss of vision. Here, we detail an organ culture open globe injury model. With this model, potential therapeutics for stabilizing these injuries can be properly evaluated.

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and ...

Biomechanical Characterization of Porcine Ocular Lens with Automated Compression

Automated Compression Testing of the Ocular Lens

The biomechanical properties of the ocular lens are essential to its function as a variable power optical element. These properties change dramatically with age in the human lens, resulting in a loss of near vision called presbyopia. However, the mechanisms of these changes remain unknown. Lens compression offers a relatively simple method for assessing the lens' biomechanical stiffness in a qualitative sense and, when coupled with appropriate analytical techniques, can help quantify biomechanical properties. A variety of lens compression tests have been performed to date, including both manual and automated, but these methods inconsistently apply key aspects of biomechanical testing such as preconditioning, loading rates, and time between measurements. This paper describes a fully automated lens compression test wherein a motorized stage is synchronized with a camera to capture the force, displacement, and shape of the lens throughout a preprogrammed loading protocol. A characteristic elastic modulus may then be calculated from these data. While demonstrated here using porcine lenses, the approach is appropriate for the compression of lenses of any species.

Author Spotlight: Advancing Understanding of Age-Related Lens Stiffness Changes 

We present an automated method for characterizing the effective elastic modulus of an ocular lens using a compression test.

The biomechanical properties of the ocular lens are essential to its function as a variable power optical element. These properties change dramatically ...

Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the optical breakdown at the laser focus. Acoustic shock waves and cavitation bubbles are generated, causing tissue rupture. Bubble sizes and pressure amplitudes vary with pulse energy and position of the focal point. In this study, enucleated porcine eyes were positioned in front of a commercially available Nd:YAG laser. Variable pulse energies as well as different positions of the focal spots posterior to the cornea were tested. Resulting lesions were evaluated by two-photon microscopy and histology to determine the best parameters for an exclusive detachment of corneal endothelial cells (CEC) with minimum collateral damage. The advantages of this method are the precise ablation of CEC, reduced collateral damage, and above all, the non-contact treatment.

Here, we present a protocol to detach corneal endothelial cells (CEC) from Descemet&#8217;s membrane (DM) using a neodymium:YAG (Nd:YAG) laser as an ex vivo disease model for bullous keratopathy (BK).

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the ...

Medicine

Microfabrication of Implantable Optics Integrated in a Microstructured Imaging Window for Advanced In Vivo Imaging

In the context of biomaterials and drug testing in animal models, this study presents a streamlined protocol for fabricating a novel implantable integrated imaging window. The micro-device comprises a sophisticated system of microlenses coupled with micro-scaffolds specifically designed for in vivo quantification of the immune response using advanced non-linear excitation microscopy. The protocol is based on two-photon polymerization (2PP) of the biocompatible photoresist SZ2080, which enables the fabrication of micro-scaffolds and micro-lenses in a continuous sequence to enhance manufacturing efficiency and precision. To further improve speed, accuracy, and structural integrity, a hybrid optics fabrication approach was implemented, involving the 2PP of the microlens outer shell followed by UV bulk crosslinking of the inner core. This innovative technique optimizes the optical properties of the microlenses while streamlining the production process. The resulting micro-device demonstrates high reproducibility and mechanical stability, making it an effective method for prototyping microscale optical systems for a range of biomedical applications.

This protocol describes the fabrication of an implantable integrated imaging window using 3D laser printing. The window consists of a system of microlenses coupled with micro-scaffolds. The method involves two-photon polymerization (2PP) of the biocompatible photoresist SZ2080 in a continuous sequence, optimizing manufacturing efficiency and alignment between the different components.

In the context of biomaterials and drug testing in animal models, this study presents a streamlined protocol for fabricating a novel implantable ...

Organ Culture System for Assessing the Toxicity of Intraocular Treatment Excipients and Pharmaceuticals

As the leading cause of blindness, cataracts are a significant burden for the tens of millions of people affected globally by this condition. Chemical exposures, among other environmental factors, are an established cause of cataracts. Ocular toxicity testing can assess whether pharmaceuticals and their components may contribute to lens damage that may lead to cataracts or aid the treatment of cataracts. 
In vitro studies and in vivo animal testing can be used for assessing the safety of chemicals prior to clinical studies. The Draize test-the current in vivo standard for ocular toxicity and irritancy testing-has been criticized for lack of sensitivity and objective measurements of determining ocular toxicity. In vitro cell-based assays are limited as cell cultures cannot appropriately model an intact functional lens. 
The method described here is a sensitive in vitro alternative to animal testing, designed to evaluate the response of the intact bovine lens to treatment at both the cellular activity level and for overall refractive performance. The non-toxic reagent resazurin is metabolized in proportion to the level of cell activity. The lens laser-scanner assay measures the ability of the lens to refract incident beams of light to a single point with minimal error, directly relevant to its natural function. The method may be used to determine both acute and delayed changes in the lens, as well as the recovery of the lens from chemical or environmental exposures.

The goal of this protocol is to evaluate changes in metabolic activity and refractive function of the lens in response to experimental treatment.

As the leading cause of blindness, cataracts are a significant burden for the tens of millions of people affected globally by this condition. Chemical ...

Biology

A High-Throughput Platform for Culture and 3D Imaging of Organoids

The characterization of a large number of three-dimensional (3D) organotypic cultures (organoids) at different resolution scales is currently limited by standard imaging approaches. This protocol describes a way to prepare microfabricated organoid culture chips, which enable multiscale, 3D live imaging on a user-friendly instrument requiring minimal manipulations and capable of up to 300 organoids/h imaging throughput. These culture chips are compatible with both air and immersion objectives (air, water, oil, and silicone) and a wide range of common microscopes (e.g., spinning disk, point scanner confocal, wide field, and brightfield). Moreover, they can be used with light-sheet modalities such as the single-objective, single-plane illumination microscopy (SPIM) technology (soSPIM). 
The protocol described here gives detailed steps for the preparation of the microfabricated culture chips and the culture and staining of organoids. Only a short length of time is required to become familiar with, and consumables and equipment can be easily found in normal biolabs. Here, the 3D imaging capabilities will be demonstrated only with commercial standard microscopes (e.g., spinning disk for 3D reconstruction and wide field microscopy for routine monitoring).

This paper presents a fabrication protocol for a new type of culture substrate with hundreds of microcontainers per mm2, in which organoids can be cultured and observed using high-resolution microscopy. The cell seeding and immunostaining protocols are also detailed.

The characterization of a large number of three-dimensional (3D) organotypic cultures (organoids) at different resolution scales is currently limited by ...

Cultivation and Characterization of Lens Epithelial Cells

Optimizing Mouse Primary Lens Epithelial Cell Culture: A Comprehensive Guide to Trypsinization

Lens epithelial cells (LECs) play multiple important roles in maintaining the homeostasis and normal function of the lens. LECs determine lens growth, development, size, and transparency. Conversely, dysfunctional LECs can lead to cataract formation and posterior capsule opacification (PCO). Consequently, establishing a robust primary LEC culture system is important to researchers engaged in lens development, biochemistry, cataract therapeutics, and PCO prevention. However, cultivating primary LECs has long presented challenges due to their limited availability, slow proliferation rate, and delicate nature. 
This study addresses these hurdles by presenting a comprehensive protocol for primary LEC culture. The protocol encompasses essential steps such as the formulation of an optimized culture medium, precise isolation of lens capsules, trypsinization techniques, subculture procedures, harvest protocols, and guidelines for storage and shipment. Throughout the culture process, cell morphology was monitored using phase-contrast microscopy. 
To confirm the authenticity of the cultured LECs, immunofluorescence assays were conducted to detect the presence and subcellular distribution of critical lens proteins, namely &#945;A- and &#947;-crystallins. This detailed protocol equips researchers with a valuable resource for cultivating and characterizing primary LECs, enabling advancements in our comprehension of lens biology and the development of therapeutic strategies for lens-related disorders.

Author Spotlight: Unlocking the Secrets of Cataracts &#8211; Investigating Redox Repair Enzymes in Lens Epithelial Cells

This manuscript outlines a detailed video protocol for culturing primary lens epithelial cells (LECs), aiming to improve reproducibility and aid research in cataracts and posterior capsule opacification (PCO). It offers step-by-step instructions on lens dissection, LECs isolation, and validation, serving as a valuable guide, especially for newcomers in the field.

Lens epithelial cells (LECs) play multiple important roles in maintaining the homeostasis and normal function of the lens. LECs determine lens growth, ...

Development of an In Vitro Ocular Platform to Test Contact Lenses

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