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

Published: January 07, 2022

doi:

Jordan Rossy, David J. McCanna, Bernard Fresco, Jacob G. Sivak

¹School of Optometry and Vision Science,University of Waterloo, ²Centre for Ocular Research & Education, School of Optometry and Vision Science,University of Waterloo, ³Tonomed Inc

Summary

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

Abstract

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.

Introduction

Affecting over 20 million people, cataracts are the most prevalent cause of blindness worldwide¹^,². Cataracts are most commonly due to age-related changes in the lens but are also induced from trauma, genetic conditions, disease, or toxic exposures². Currently, treatment involves surgical intervention to replace the lens, an expensive and invasive procedure accessible mainly to those in developed countries. The extensive burden of cataract has directed decades of research towards cataract prevention and the development of non-surgical treatment. In both cases, the importance of preclinical testing for toxicity, efficacy, and pharmacokinetics of ophthalmic drugs is paramount. This process of drug development relies heavily on the information provided by studies performed in animals.

The current standard for ocular toxicity testing in vivo is the Draize test, involving the delivery of a test compound to the conjunctival sac of a live animal. The test has been significantly criticized, particularly concerning animal ethics, subjectivity, poor repeatability, and variability³. Additionally, there is no component of the Draize test that directly monitors the effects of test substances on the lens. Considerable effort has been invested in developing alternative in vitro models⁴. However, none have been sufficiently validated to replace the Draize test⁵. Similarly, many of these models face limitations with respect to the direct application to cataracts and other complex pathologies⁶. For example, methods grading lens transparency when placed over a grid are inherently subjective⁷. Cell culture studies are reliable and highly utilized, though cell monolayer characteristics may diverge from primary tissue culture⁸.

Whole lenses can be dissected from the eyes of animals and cultured to maintain their original structure and function. One assay that is useful for assessing lens function while maintaining the organ's condition is the lens laser-scanner assay involving a scanner developed at the University of Waterloo in Canada. The assay is a scanning system that uses a series of laser projections to measure the optical quality or refractive performance of the lens. Lenses are scanned in their custom two-segment culture chambers, allowing beams to pass from below through the lens (Figure 1A). A camera fixed inside the scanner captures the image of the laser passing through the lens at numerous points. The scanner software computes the distance behind the lens at which it intersects with a central axis (back vertex distance, BVD), producing a series of measurements that indicate how consistently the lens focuses light to a single point (Figure 1).

The cellular properties of the lens, such as the tight and ordered arrangement of its cells, help maintain transparency and minimize scatter so that the lens can functionally focus light⁹. This measure can be used to interpret how significantly a chemical disrupts the essential structure of the lens, such as the gradient refractive index, and how much function is compromised because of the induced opacities. Other studies that have followed the response of cultured lenses and lens vesicles suggest that light scatter is a product of structural changes, as compared to metabolic changes, and that disruptions to lens lipids and proteins may affect the refractive index and consequently increase scatter¹⁰^,¹¹.

The lens laser-scanner can be used in conjunction with metabolic reagents in assays to determine biochemical measures of cell toxicity. Resazurin is a non-toxic chemical reagent metabolized by active cells, producing a reduced product (resorufin) with a measurable fluorescence¹². The lens is largely devoid of organelles, except the metabolically active mitochondria concentrated within the anterior epithelium and superficial cortical fiber cells, fulfilling lens energy requirements¹³^,¹⁴. Damage to the lens at the cellular level may disrupt metabolism and often precedes the onset of pathogenic structural changes and cataract¹⁵.

The purpose of this method is to evaluate the effect of xenobiotic and environmental exposures on the lens, which may contribute to cataract development. The protocol involves two assays to evaluate the effect of a treatment using the cultured bovine lens. The advantage of this approach is that it provides both a cellular and functional evaluation of how the lens as a primary tissue responds to treatment. It is a sensitive and objective evaluation of the lens as compared to other common methods¹⁶^,¹⁷^,¹⁸.

The model has been successfully used to evaluate the effects of various exposures, including surfactants, consumer products, alcohols, and ultraviolet radiation¹⁷^,¹⁹^,²⁰. Changes in optical quality are consistently present in cultured lenses as a response to toxic exposure²¹. The ability of this method to maintain long-term lens culture is well-suited for monitoring the potentially delayed effect of a compound, and the recovery of the lens from induced damage or cataract²²^,²³. Results produced from the application of this protocol can be used to reduce dependence on animal testing in the development of ophthalmic products.

Protocol

All experimental protocols were carried out in compliance with the University of Waterloo ethics policies for research using animal tissue. The bovine eyes for the current study were abattoir-provided, obtained from non-dairy cows within a few hours of death, and were dissected immediately, a process that takes up to 8 h from obtaining the eyes. Eyes should be dissected immediately to preserve sterility and dissection quality. The culture medium is prepared to a pH of 7.4 and sterile-filtered prior to supplementation wit…

Representative Results

Figure 2 and Figure 3 (n = 6) demonstrate the results of a study testing the effect of chemical treatment (lanosterol) on the bovine lens. Lanosterol is a naturally occurring sterol in the lens that once showed promising results as a potential pharmaceutical intervention for cataracts25, although this has yet to be proven26. The study design included a medium and vehicle control for the compound. There was no signi…

Discussion

The purpose of this protocol is to directly evaluate the effects of chemicals or environmental exposures on the lens in primary tissue culture. First, lenses are dissected and scanned for optical quality. Prevention of contamination and ensuring dissection quality are critical. Lenses are scanned at periodic intervals to continuously monitor changes in refractive function with respect to the control group or preexposure condition. The metabolic activity assay represents an endpoint to determine whether the exposures have…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Thanks to the Natural Sciences and Engineering Research Council (NSERC) and the Canadian Optometric Education Trust Fund (COETF) for the funds for this project.

Materials

(2-Hydroxypropyl)-β-cyclodextrin	Sigma-Aldrich	H107	Powder
1 L bottle-top filtration system	VWR	97066-204	Full Assembly, bottle-top, 0.2 μm
100 mm Petri dish	VWR	89022-320	Slippable, media saver style, sterile
12 well-plate	Corning	353043	Sterile, clear-bottom
35 mm petri dish	VWR	25373-041	Falcon disposable petri dishes, sterile, Corning
96 well-plate	VWR	29442-072	Sterile, clear-bottom
Alamar blue (resazurin)	Fischer Scientific	DAL1100	Molecular Probes cell viability reagent
Benzalkonium chloride solution	Sigma-Aldrich	63249	50% in H₂0
Biosafety cabinet
Cytation 5 plate reader	BioTek	CYT5MPV	Cell imaging multi-mode reader
Fetal bovine serum	ThermoFischer Scientific	12484028	Qualified, heat inactivated, Canada
HEPES	Sigma-Aldrich	H3375	For cell culture, powder
Incubator
Lanosterol	Sigma-Aldrich	L5768	≥93%, powder
L-glutamine	Sigma-Aldrich		For cell culture, powder
Medium (M-199)	Sigma-Aldrich	M3769	Modified, with Earle′s salts, without L-glutamine, sodium bicarbonate, and phenol red, powder, suitable for cell culture
Pasteur pipettes			5 3/4'', with and without cotton
Penicillin-Streptomycin	ThermoFischer Scientific	15140122	Liquid (10,000 U/mL)
Phospate buffer saline (PBS)			liquid, sterile, suitable for cell culture
Pipette tips (100 µL, 1,000 µL, 5,000 µL)	VWR		Sterile
ScanTox (lens laser-scanner)	Specially developed in-house	N/A	Scans lens with a laser to determine lens optical quality
ScanTox culture chamber	Specially developed in-house	N/A	Holds bovine lens in place during testing and culturing
Sodium bicarbonate	Sigma-Aldrich	S5761	For cell culture, powder
Sodium hydroxide	Sigma-Aldrich	S2770	1.0 N, BioReagent, suitable for cell culture

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