Using the Cytosensor Microphysiometer to Assess Ocular Toxicity

Jennifer R. Nash1, Greg Mun1, Hans A. Raabe1, Rodger Curren1

1 Institute for In Vitro Sciences, Inc., Gaithersburg, Maryland
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 1.13
DOI:  10.1002/0471140856.tx0113s61
Online Posting Date:  August, 2014
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Measuring in vitro cytotoxicity is one method currently used to estimate damage to the eye after chemical exposure. The Cytosensor Microphysiometer method evaluates cytotoxicity by measuring the test material–induced reduction in the metabolic rate of L929 cells. Changes in metabolic rate are measured indirectly as a function of changes in the extracellular acidification rate of the cells. During exposure to increasing concentrations of a cytotoxic material, there is a decrease in the release of acid byproducts into the surrounding medium as the cells die. These acidic metabolic byproducts cause a measurable change in the pH of a lightly buffered medium, which can be measured by the Cytosensor Microphysiometer. The change in the pH of the medium over time is then converted into a metabolic rate estimate for the cells. The endpoint measurement from the assay is the metabolic rate decline of 50%, the MRD50 value (in units of mg/ml). Curr. Protoc. Toxicol. 61:1.13.1‐1.13.11. © 2014 by John Wiley & Sons, Inc.

Keywords: eye irritation; ocular irritation; in vitro cytotoxicity; Cytosensor Microphysiometer; metabolic rate

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1:

  • L929 mouse fibroblasts (ATCC #CCL‐1, NCTC Clone 929)
  • Growth medium (see recipe)
  • Phosphate‐buffered saline (PBS; see recipe)
  • 0.05% trypsin (see recipe)
  • Seeding medium (see recipe)
  • Low‐buffered medium (see recipe)
  • Sterilant solution (see recipe)
  • Sterile H 2O
  • Test article(s)
  • Positive control: 10% SDS (see recipe; use dilutions as shown in Table 1.13.1)
  • 75‐cm2 (T‐75) tissue culture flasks
  • 50‐ml conical centrifuge tubes (e.g., BD Falcon)
  • Coulter counter or hemacytometer for counting cells ( appendix 3B)
  • 0.3 µm pore size, 12 mm diameter capsule cups (transwells; Corning, cat. no. 3462)
  • 12‐well tissue culture plates
  • Cytosensor Microphysiometer (no longer available from manufacturer; can be found on Internet auction sites or through used equipment dealers)
  • Computer running Cytosoft software (no longer available from manufacturer; can be found on Internet auction sites or through used equipment dealers)
  • Debubbler membranes (no longer available from manufacturer; can be found on Internet auction sites or through used equipment dealers)
  • KCl reference electrode (Thermo Scientific, cat. no. 900004; requires fill solution consisting of 2 M KCl with Ag, also from Thermo Scientific)
  • Spacers (Molecular Devices)
  • Forceps
  • 3‐ml syringes
  • Additional reagents and equipment for cell culture, including trypsinization and cell counting ( appendix 3B)
Table 1.3.1   MaterialsDosing Concentrations

Dilution no. Test article concentration Positive control concentration
A (not dosed) 10 mg/ml
1 10 mg/ml 3.0 mg/ml
2 3.33 mg/ml 1.0 mg/ml
3 1.11 mg/ml 0.3 mg/ml
4 0.370 mg/ml 0.1 mg/ml
5 0.123 mg/ml 0.03 mg/ml
6 0.0412 mg/ml 0.001 mg/ml
7 0.0137 mg/ml 0.003 mg/ml

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Literature Cited

Literature Cited
  Bagley, D.M., Bruner, L.H., DeSilva, O., Cottin, M., O'Brien, K.A.F., Uttley, M., and Walker, A.P. 1992. An evaluation of five potential alternatives in vitro to the rabbit eye irritation test in vivo. Toxicol. In Vitro 6:275‐284.
  Balls, M., Botham, P.A., Bruner, L.H., and Spielmann, H. 1995. The EC/HO international validation study on alternatives to the Draize eye irritation test. Toxicol. In Vitro 9:871‐929.
  Bruner, L.H., Miller, K.M., Owicki, J.C., Parce, J.W., and Muir, V.C. 1991. Testing ocular irritancy in vitro with the silicon microphysiometer. Toxicol. In Vitro 5:277‐284.
  Curren, R.D., Sizemore, A.S., Nash, J., Mun, G., Ulrey, A., and Harbell, J.W. 2008. Background review document of existing methods for eye irritation testing: Silicon microphysiometer and cytosensor microphysiometer.‐ecvam/validation‐regulatory‐acceptance/docs‐eye‐irritation/ECVAM‐CMBRD‐Aug08cleaned.pdf (last accessed 25 April 2014).
  ECVAM. 2010. Statement On The Scientific Validity Of Cytotoxicity/Cell‐Function Based In Vitro Assays For Eye Irritation Testing.‐ecvam/publication/ESAC31_CBA_eye‐irritation_20091005.pdf/view (last accessed 25 April 2014).
  Gettings, S.D., Lordo, R.A., Hintze, K.L., Bagley, D.M., Casterton, P.L., Chudkowski, M., Curren, R.D., Demetrulias, L., Dipasquale, L.C., Earl, L.K., Feder, P.I., Galli, L., Glaza, S.M., Gordon, V.C., Janus, J., Kurtz, P.J., Marenus, K.D., Moral, J., Paper, W.J.W., Renskers, K.J., Rheins, L.A., Roddy, M.T., Rozen, M.G., Tedeschi, J.P., and Zyracki, J. 1996. The CFTA evaluation of alternatives program: An evaluation of in vitro alternatives to the Draize primary eye irritation test. (Phase III) surfactant‐based formulations. Food Chem. Toxicol. 34:79‐117.
  Harbell, J.W., Osborne, R., Carr, G.J., and Peterson, A. 1999. Assessment of the cytosensor microphysiometer assay in the COLIPA in vitro eye irritation validation study. Toxicol. In Vitro 13:313‐323.
  Hartung, T., Bruner, L., Curren, R., Eskes, C., Goldberg, A., McNamee, P., Scott, L., and Zuang, V. 2010 First alternative method validated by a retrospective weight‐of‐evidence approach to replace the Draize eye test for the identification of non‐irritant substances for a defined applicability domain. ALTEX 27:43‐51.
  Parce, J.W., Owicki, J.C., Kercso, K.M., Sigal, G.B., Wada, H.G., Muir, V.C., Bousse, L.J., Ross, K.L., Sikic, B.I., and McConnell, H.M. 1989. Detection of cell‐affecting agents with a silicon biosensor. Science 246:243‐247.
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