Name: development of an in vitro ocular platform to test contact lenses
Uploaded: 2016-04-06T15:00:00
Description: Watch this Scientific Journal Video about Development of an In Vitro Ocular Platform to Test Contact Lenses at JoVE.com

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. Desi...

<ol>
	<li>Phan, C. M., Subbaraman, L. N., Jones, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+drug+release+of+natamycin+from+beta-cyclodextrin+and+2-hydroxypropyl-beta-cyclodextrin-functionalized+contact+lens+materials.">In vitro drug release of natamycin from beta-cyclodextrin and 2-hydroxypropyl-beta-cyclodextrin-functionalized contact lens materials.</a> J Biomater Sci Polym Ed. 25, 1907-1919 (2014).</li><li>Peng, C. C., Kim, J., Chauhan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extended+delivery+of+hydrophilic+drugs+from+silicone-hydrogel+contact+lenses+containing+vitamin+E+diffusion+barriers.">Extended delivery of hydrophilic drugs from silicone-hydrogel contact lenses containing vitamin E diffusion barriers.</a> Biomaterials. 31, 4032-4047 (2010).</li><li>Hui, A., Willcox, M., Jones, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+evaluation+of+novel+ciprofloxacin-releasing+silicone+hydrogel+contact+lenses.">In vitro and in vivo evaluation of novel ciprofloxacin-releasing silicone hydrogel contact lenses.</a> Invest Ophthalmol Vis Sci. 55, 4896-4904 (2014).</li><li>Boone, A., Hui, A., Jones, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake+and+release+of+dexamethasone+phosphate+from+silicone+hydrogel+and+group+I,+II,+and+IV+hydrogel+contact+lenses.">Uptake and release of dexamethasone phosphate from silicone hydrogel and group I, II, and IV hydrogel contact lenses.</a> Eye Contact Lens. 35, 260-267 (2009).</li><li>Lorentz, H., Heynen, M., Trieu, D., Hagedorn, S. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cornea. , (2002).</li><li>Parekh, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+technique+for+in+situ+excision+of+cornea+and+evisceration+of+retinal+tissue+from+human+ocular+globe.">A simplified technique for in situ excision of cornea and evisceration of retinal tissue from human ocular globe.</a> Journal of visualized experiments : JoVE. , e3765 (2012).</li><li>Way, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gear+and+pinion.">Gear and pinion.</a> US patent. , (1942).</li><li>Lorentz, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contact+lens+physical+properties+and+lipid+deposition+in+a+novel+characterized+artificial+tear+solution.">Contact lens physical properties and lipid deposition in a novel characterized artificial tear solution.</a> Molecular vision. 17, 3392-3405 (2011).</li><li>Furukawa, R. E., Polse, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+tear+flow+accompanying+aging.">Changes in tear flow accompanying aging.</a> American journal of optometry and physiological optics. 55, 69-74 (1978).</li><li>Bajgrowicz, M., Phan, C. M., Subbaraman, L., Jones, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Release+of+ciprofloxacin+and+moxifloxacin+from+daily+disposable+contact+lenses+from+an+in+vitro+eye+model.">Release of ciprofloxacin and moxifloxacin from daily disposable contact lenses from an in vitro eye model.</a> Invest Ophthalmol Vis Sci. , (2015).</li><li>Luensmann, D., Zhang, F., Subbaraman, L., Sheardown, H., Jones, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+lysozyme+sorption+to+conventional+and+silicone+hydrogel+contact+lenses+using+confocal+microscopy.">Localization of lysozyme sorption to conventional and silicone hydrogel contact lenses using confocal microscopy.</a> Current eye research. 34, 683-697 (2009).</li><li>Tieppo, A., Pate, K. M., Byrne, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+controlled+release+of+an+anti-inflammatory+from+daily+disposable+therapeutic+contact+lenses+under+physiological+ocular+tear+flow.">In vitro controlled release of an anti-inflammatory from daily disposable therapeutic contact lenses under physiological ocular tear flow.</a> Eur J Pharm Biopharm. 81, 170-177 (2012).</li><li>Ali, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zero-order+therapeutic+release+from+imprinted+hydrogel+contact+lenses+within+in+vitro+physiological+ocular+tear+flow.">Zero-order therapeutic release from imprinted hydrogel contact lenses within in vitro physiological ocular tear flow.</a> J Control Release. 124, 154-162 (2007).</li><li>White, C. J., McBride, M. K., Pate, K. M., Tieppo, A., Byrne, M. 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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...

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 border="0" cellpadding="0" cellspacing="0" width="1129">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">Arduino Uno R3 (Atmega328 - assembled)</td>
			<td style="width:125px;">Adafruit</td>
			<td style="width:451px;">50</td>
			<td style="width:273px;">Board</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Stepper motor</td>
			<td style="width:125px;">Adafruit</td>
			<td style="width:451px;">324</td>
			<td>Motor and Motor shield</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Equal Leg Coupler 1.6mm 1/16&#34;</td>
			<td style="width:125px;">VWR</td>
			<td style="width:451px;">CA11009-280</td>
			<td>50 pcs of tube connector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Tubing PT/SIL 1/16&#34;x1/8&#34;</td>
			<td style="width:125px;">VWR</td>
			<td style="width:451px;">16211-316</td>
			<td>Case of 50feet</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">PDMS</td>
			<td style="width:125px;">Dow Corning</td>
			<td style="width:451px;">Sylgard 184 Solar Cell Encapsulation</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Agarose, Type 1-A, low EEO</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:451px;">A0169-25G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">PHD UltraTM</td>
			<td style="width:125px;">Harvard Apparatus</td>
			<td style="width:451px;">703006</td>
			<td>MicroFluidic Pump</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">Bovine cornea</td>
			<td style="width:125px;">Cargill, Guelph/ON</td>
			<td style="width:451px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">Soldidworks</td>
			<td style="width:125px;">Dassault Systemes</td>
			<td style="width:451px;"></td>
			<td>Software</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:281px;">3-D printing</td>
			<td style="width:125px;">University of Waterloo - 3D Print Centre</td>
			<td style="width:451px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">Dissection tools</td>
			<td style="width:125px;">Fine Science Tools</td>
			<td style="width:451px;"></td>
			<td>General dissection tools</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Medium 199</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:451px;"></td>
			<td>Culture medium storage for cornea</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Fetal bovine serum</td>
			<td style="width:125px;">Thermo Fisher</td>
			<td style="width:451px;"></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 ...

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 ...

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.

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In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

Rodent Behavioral Testing to Assess Functional Deficits Caused by Microelectrode Implantation in the Rat Motor Cortex

Medical devices implanted in the brain hold tremendous potential. As part of a Brain Machine Interface (BMI) system, intracortical microelectrodes ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...