Validation of eGFP fluorescence intensity for testing in vitro cytotoxicity according to ISO 10993‐5

Abstract

ISO 10993-5 provides one of the accepted standards for testing the biotoxicity of new materials. All of the recommended test procedures rely upon the uptake or metabolism of dye by living cells. Results of direct contact tests can be potentially compromised by interaction or adsorption of the dye or its metabolic products. Therefore, the aim of the current study was to validate the use of the eGFP signal of transfected NIH-3T3 fibroblasts with the results of the MTT test in order to provide a test procedure that is very close to the ISO 10993-5 but has the advantage of not relying on the addition of dye. Our tests show that the MTT assay detects cytotoxicity in the eGFP NIH-3T3 cells at least as well as in the L929 cells. To facilitate the validation, we chose to integrate the fluorescence measurements into the MTT test procedure. To that end, an additional washing step was introduced. Additionally, medium without phenol red was used, resulting in a very high correlation of both measurements. Without these modifications, the fluorescence test was comparable to the MTT test in its ability to detect the cytotoxic potential of substances; however, it did result in slightly elevated IC50 concentrations. As the results of both tests correlated highly, measurement of the eGFP signal appears to present a reliable tool for detecting cytotoxicity of materials in line with the ISO 10993-5 norm with the advantage of avoiding the addition of dyes and the subsequent potential interaction with test materials. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 715-722, 2017.

In vitro and in vivo biocompatibility of Ti-6Al-4V titanium alloy and UHMWPE polymer for total hip replacement

“…The results showed that both materials did not affect 3T3 cell morphology or growth. Although there are new suggested methods to assess cytotoxicity of materials (Miller et al, 2015), the cytotoxicity assay based on 3T3 cell proliferation is still considered a traditional method, and entails measuring neutral red uptake (Borenfreund et al, 1988;Borenfreund and Puerner, 1985;Repetto et al, 2008).…”

Anti-Tumor Efficiency of Perillylalcohol/β-Cyclodextrin Inclusion Complexes in a Sarcoma S180-Induced Mice Model

“…Viability assay was performed with human fibroblast line L929 based on the ISO 10993–5 [ 36 ]. L929 cells (ATCC, LGC Standards S.L.U., Barcelona, Spain) were seeded in 96-well culture plates (2 × 10 4 cells/well) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) composed of NaHCO 3 (1.2 g/L), ampicillin (0.025 g/L), streptomycin (0.1 g/L), supplemented with 10% fetal bovine serum (SFB).…”

Mesoporous Silica-Bioglass Composite Pellets as Bone Drug Delivery System with Mineralization Potential

“…This test is related to direct interaction between investigated material and cell monolayer. To avoid false-negative results due to the adsorption of dye (such as MTT, XTT, WST) onto the surface of the pellets, we decided to perform an eGFP fluorescence-based assay which is a reliable and sensitive tool for testing the cytotoxicity of materials [ 69 ]. Therefore, at first, osteoblasts were transfected for stable production of enhanced GFP.…”