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

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 worldwide^1,2. 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 preclini....

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 with FBS²¹. All procedures are carried out under sterile conditions, with material and equipment sources listed in the Ta....

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
(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