June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Using replacement methods to live animal testing for evaluating the toxicity/fixation potential of candidate therapeutic tissue cross-linking agents
Author Affiliations & Notes
  • Su-Young Kim
    Ophthalmology, Columbia University Medical Center, New York, NY
  • Brendan Roach
    Biomedical Engineering, Columbia University, New York, NY
  • Natasha Babar
    Ophthalmology, Columbia University Medical Center, New York, NY
  • Anna Takaoka
    Ophthalmology, Columbia University Medical Center, New York, NY
  • Clark Hung
    Biomedical Engineering, Columbia University, New York, NY
  • Stephen Trokel
    Ophthalmology, Columbia University Medical Center, New York, NY
  • David C. Paik
    Ophthalmology, Columbia University Medical Center, New York, NY
  • Footnotes
    Commercial Relationships Su-Young Kim, None; Brendan Roach, None; Natasha Babar, None; Anna Takaoka, None; Clark Hung, None; Stephen Trokel, None; David C. Paik, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 1127. doi:
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      Su-Young Kim, Brendan Roach, Natasha Babar, Anna Takaoka, Clark Hung, Stephen Trokel, David C. Paik; Using replacement methods to live animal testing for evaluating the toxicity/fixation potential of candidate therapeutic tissue cross-linking agents. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1127.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: As the field of corneal cross-linking (CXL) and therapeutic tissue cross-linking (TXL) in other tissues (such as sclera) moves forward, understanding the relationship or balance between the ability to induce mechanical tissue changes and killing cells becomes relevant. This could be particularly important as we pursue refinements in either chemical or photochemical approaches. Thus, the present study was undertaken in order to develop a potential replacement method for live animal testing when evaluating such approaches.

Methods: Organ cultured bovine cartilage explants and fresh cadaveric rabbit corneas in an ex vivo corneal crosslinking simulation set up was used. The tissue was exposed to the formaldehyde releasing agent (FAR) diazolidinyl urea (DAU) under continuous exposure (22, 220, 2200 uM) in low glucose explant growth medium at pH 7.2 for 3 to 7 days for cartilage; and for 30min (1.8mM epithelium-off and 8.9mM epithelium-on) at pH 8.5 for cornea. As a comparison, the same ex vivo system was used to conduct riboflavin-mediated photochemical CXL of cornea. Post-exposure, the tissue viability was assessed using a LIVE/DEAD Viability/Cytotoxicity Kit (Life Technologies) and imaged on an inverted confocal microscope. Thermal transition temperature (Tm) was determined by differential scanning calorimetry.

Results: Concentration dependent effects in cell viability were noted. Lower concentrations and shorter exposure times were associated with lower cell toxicity. In the cartilage, DAU was cytotoxic at 2200uM, showed a time dependent effect at 220uM, and was well tolerated at 22uM. In corneal tissue that showed shifts in Tm by DSC, we were able to visualize both live and dead cells in both the endothelial and stromal regions.

Conclusions: These methods could be very helpful in refining our current approaches to tissue cross-linking therapy and are a possible “replacement” method to live animal testing. These methods will be further developed for the purpose of evaluating the “toxicity/fixation balance” of therapeutic tissue cross-linking approaches.

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