The doctoral dissertations of the former Helsinki University of Technology (TKK) and Aalto University Schools of Technology (CHEM, ELEC, ENG, SCI) published in electronic format are available in the electronic publications archive of Aalto University - Aaltodoc.

Corneal Epithelial and Retinal Pigment Epithelial Cell Culture Assays as Potential Alternatives to Animal Experimentation for the Evaluation of Ocular Toxicity

Anne Huhtala

Dissertation for the degree of Doctor of Science in Technology to be presented with due permission of the Department of Chemical Technology for public examination and debate in Auditorium KE 2 (Komppa Auditorium) at Helsinki University of Technology (Espoo, Finland) on the 10th of October, 2003, at 12 o'clock noon.

Overview in PDF format (ISBN 951-22-6734-9)   [8598 KB]
Dissertation is also available in print (ISBN 951-22-6733-0)


The widely criticized Draize rabbit test is the only eye toxicity test officially accepted worldwide for regulatory purposes in the classification of slightly and moderately irritating chemicals. Today, there are no in vitro alternatives that could be used as a complete replacement for the Draize eye test. Moreover, in the current OECD guidelines, there are no tests for retinal toxicity, not even in vivo.

This study was undertaken to develop corneal epithelial and retinal pigment epithelial cell culture assays as pre-screens and potential alternatives in a more comprehensive battery test for in vitro eye toxicity testing. Two basal cytotoxicity tests with effective 96-well techniques and plate readers were established to evaluate the adverse ocular effects of selected test compounds: WST-1 test as an index of mitochondrial function and cell viability/proliferation, and lactate dehydrogenase (LDH) leakage test as an index of cell membrane integrity.

The confluent and post-confluent human corneal epithelial (HCE) cell line in culture medium was found to express cornea-specific cytokeratin 3, but it also expressed simple epithelium-specific cytokeratins 7, 8, 18 and 19. In most cases in the WST-1 and LDH testing, the pre-confluent HCE cell line was as sensitive to the test compounds as the pre-confluent primary cultures of rabbit corneal epithelial epithelium. The WST-1 test appeared to be an earlier indicator of toxicity than the LDH test, which also showed great variations in the cytotoxicity results. In the HCE-WST-1 interlaboratory study, the most reproducible results were obtained when the cells were exposed to the test substances for one hour in the absence of serum. The use of the human retinal pigment epithelial cell line D407 and of pig primary retinal pigment epithelial cell cultures in the WST-1 testing also yielded comparable results.

In all cases, the use of serum in culture medium resulted in lower toxicity, and thus its use is not recommended. The assays studied, especially those based on cell lines, are reliable, transferable, easy-to-perform tests for ocular toxicity screening.

This thesis consists of an overview and of the following 5 publications:

  1. Huhtala A., Nurmi S. K., Tähti H., Salminen L., Alajuuma P., Rantala I., Helin H. and Uusitalo H., 2003. The immunohistochemical characterisation of an SV40-immortalized human corneal epithelial cell line. Alternatives to Laboratory Animals 31, pages 409-417. © 2003 by authors and © 2003 FRAME. By permission.
  2. Huhtala A., Mannerström M., Alajuuma P., Nurmi S., Toimela T., Tähti H., Salminen L. and Uusitalo H., 2002. Comparison of an immortalized human corneal epithelial cell line and rabbit corneal epithelial cell culture in cytotoxicity testing. Journal of Ocular Pharmacology and Therapeutics 18, No. 2, pages 163-175. © 2002 Mary Ann Liebert, Inc. Publishers. By permission.
  3. Huhtala A., Alajuuma P., Burgalassi S., Chetoni P., Diehl H., Engelke M., Marselos M., Monti D., Pappas P., Saettone M. F., Salminen L., Sotiropoulou M., Tähti H., Uusitalo H. and Zorn-Kruppa M., 2003. A collaborative evaluation of the cytotoxicity of two surfactants by using the human corneal epithelial cell line and the WST-1 Test. Journal of Ocular Pharmacology and Therapeutics 19, No. 1, pages 11-21. © 2003 Mary Ann Liebert, Inc. Publishers. By permission.
  4. Huhtala A., Tähti H., Salminen L. and Uusitalo H., 2002. Evaluation of adverse ocular effects of 5-fluorouracil by using human corneal epithelial cell cultures. Journal of Toxicology: Cutaneous and Ocular Toxicology 21, No. 4, pages 283-292. © 2002 Marcel Dekker, Inc. By permission.
  5. Mannerström M., Zorn-Kruppa M., Diehl H., Engelke M., Toimela T., Mäenpää H., Huhtala A., Uusitalo H., Salminen L., Pappas P., Marselos M., Mäntylä M., Mäntylä E. and Tähti H., 2002. Evaluation of the cytotoxicity of selected systemic and intravitreally dosed drugs in the cultures of human retinal pigment epithelial cell line and of pig primary retinal pigment epithelial cells. Toxicology In Vitro 16, pages 193-200. © 2002 Elsevier Science. By permission.

Keywords: draize eye test, eye irritation, ocular toxicity, alternative methods, in vitro test, cytotoxicity, cell culture, corneal epithelial cell, retinal pigment epithelial cell

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© 2003 Helsinki University of Technology

Last update 2011-05-26