The Alchilife R& team, in collaboration with Mario Romano, MD, is one of the first laboratories that validated a direct contact method for the cytotoxicity assay performed according to the ISO 10993-5 standard on perfluorocarbons (PFCLs) for ophthalmic surgery.

Selecting a truly reliable method for the cytotoxicity evaluation of PFLCs is the key strategy to avoid serious concerns about patients’ safety. The unfortunate past has proved that the “extract dilution” method, despite being mentioned among the acceptable tests by the ISO 10993-5 standard, is not a reliable assay for medical devices containing volatile substances. Conversely, the direct contact test – which has always been the method of choice used in the Alchilife laboratories, is highly sensitive, able to detect even low cytotoxicity, and is gaining increased interest in the field of PFCL safety.

By exploiting the high-density properties of liquid PFCLs, the team of Alchilife researchers could ensure the effective direct contact between the tested perfluorocarbon and the cell substrate (i.e. the ARPE-19 and BALB 3T3 cell layer) for the whole duration of the test by careful injecting the perfluorocarbon liquid with the tip of the pipette immersed in the medium right above the cell lines: this creates a single dense bubble that gently lays on the cells due to the force of gravity while avoiding excessive pressure on the cell layer because of the sample heaviness. This step simulates what happens in the eye during the clinical use. The so-formed dense bubble avoids any sample evaporation or loss of contact of PFCL with the cells. This method allows to overcome the problem of PFCL insolubility in water, which prevents the sample from diluting into the medium – the latter may be one of the factors that impairs the reliability of the extraction method for testing the cytotoxicity of PFCL.

With the approach by Alchilife various amount of contact areas and contact times (up to 24 hours) can be successfully tested. As a result of this study, whose preliminary results were presented by Dr Romano at the 18th Euretina Congress in Vienna, the direct contact cytotoxicity test of PFCLs, which warrants covering of 59% of the cell surface and contact time of 24 h according to ISO 10993-5, was validated quantitatively and qualitatively on ARPE19 and BALB 3T3 cell lines.

References:

  • ISO 10993-5, 2009. Biological evaluation of medical devices – Part 5: Tests for in vitro cytotoxicity.
    https://www.iso.org/obp/ui/#iso:std:iso:10993:-5:en (accessed 20 July 2019)
  • Li et al. Study on the in vitro cytotoxicity testing of medical devices(Review). Biomed Rep 2015;3(5):617-620.
  • Srivastava GK et al. Comparison between direct contact and extract exposure methods for PFO cytotoxity evaluation. Sci Rep 2018;8(1):1425.
  • Romano M. Perfluorocarbons for intraocular use: cytotoxicity test validation according to the ISO 10993-5. Presentation at Euretina 2018
  • Romano MR et al. Evaluation of Cytotoxicity of Perfluorocarbons for Intraocular Use by Cytotoxicity Test In Vitro in Cell Lines and Human Donor Retina Ex Vivo. Transl Vis Sci Technol 2019; 8(5):24.