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Testing Toxicity On Primary Cultures Of Cornea Instead Of Animal Models

Several tissues make up the cornea that ranges from the epithelium to the basement membrane that in turn maintains the epithelium. This is followed by the keratocytes in the stroma that produce the collagen for repair and extracellular matrix proteins. An inner layer called Descemet’s membrane that allows the passage of drugs applied on the cornea to the next layer called the aqueous humour.

According to research in Advanced Drug Delivery Reviews by Urtti, the cornea becomes an important route by which drugs get access into the chambers of the eye. Thus, the possible toxic effects of drugs can be seen in the cells of the cornea as they are exposed to drugs.

The possible toxic effects of drugs when it comes to the eyes are studied using animal models. An example is the Draize test. This involves administering a drug or chemical under test into the lower conjunctival sac of an albino rabbit. Any changes seen in the eye such as redness of the conjunctiva, inflammation of the iris or iritis, swelling or oedema and discharge of tears are recorded. Not only does this test use animals that are inflicted with discomfort or pain but also the issue of the observations being subjective or dependant on the person who checks them. Another challenge is the difference in the biology-anatomy and physiology of human and rabbit eyes.

For assessing the biodistribution in vivo, Rönkkö and team have reported in Drug Delivery and Translational Research that a minimum of 20 animals (rabbits) are sacrificed! This is when 2 drugs are compared using at least 5-time points requiring 2 pairs of eyes per point. The graphs for observation are generated by killing the animals as sampling cannot be non-invasive.

This brings in the use of in vitro approaches to assess toxicity. This becomes pertinent given that the Cosmetics Directive 76/768/EEC bans the use of animals for testing cosmetics in the European Union. Another law (Directive 63/2010/EU) looks at replacing, reducing and refining the use of animals for research.

The testing of drugs on the eyes has also used ex vivo systems. Examples are the use of corneas isolated from bovine sources for use in bovine corneal opacity and permeability (BCOP) assays. After applying the compound of interest, effects are seen. Not only are these tests valid for a few hours but they again do not represent human eyes.

Rönkkö and team (2016) have reported several studies using primary cultures of epithelial cells of the cornea. These range from studying toxicity to uptake studies and the effect of growth factors. As primary cells do not undergo modification and hence, match the phenotype of the cornea in vivo, they can be termed as representatives of the human corneal epithelial cells.

The effect of chemical molecules being studied can be assessed on primary cultures by observing for effects such as redness or cell migration or swelling. Toxicity to cells can be checked by measuring the viability of cells using colorimetric tests or luminescence. The molecules released such as cytokines or ATP in response to toxic molecules can also be measured.

For example, Geerling and team established primary cultures of corneal epithelial cells of humans using keratinocyte serum-free medium. The article published in Investigative Ophthalmology & Visual Science reported the testing of the cultures with tear substitutes such as hydroxypropylmethylcellulose with the use of ATP and luminescence.

Apart from discovering new molecules to be used as therapeutic agents, research also aims at lowering the use of animals for testing. Primary cultures are close to in vivo systems making them suitable to assess drugs under test thus aiding research invaluably.


Urtti A. Challenges and obstacles of ocular pharmacokinetics and drug delivery. Advanced Drug Delivery Reviews 2006; 58(11):1131–5.

Wilson SL, Ahearne M, Hopkinson A. An overview of current techniques for ocular toxicity testing. Toxicology 2015; 327:32–46.

Rönkkö S, Vellonen K, Järvinen K. et al. Human corneal cell culture models for drug toxicity studies. Drug Delivery and Translational Research 2016; 6, 660–675.

Geerling G, Daniels JT, Dart JK, Cree IA, Khaw PT. Toxicity of natural tear substitutes in a fully defined culture model of human corneal epithelial cells. Investigative Ophthalmology & Visual Science 2001; 42(5):948–56.

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