Details
| Original language | English |
|---|---|
| Pages (from-to) | 715-722 |
| Number of pages | 8 |
| Journal | Journal of Biomedical Materials Research - Part B Applied Biomaterials |
| Volume | 105 |
| Issue number | 4 |
| Publication status | Published - 24 Dec 2015 |
| Externally published | Yes |
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.
Keywords
- eGFP fluorescence, implant material, in vitro cytotoxicity test, ISO 10993-5
ASJC Scopus subject areas
- Materials Science(all)
- Biomaterials
- Engineering(all)
- Biomedical Engineering
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In: Journal of Biomedical Materials Research - Part B Applied Biomaterials, Vol. 105, No. 4, 24.12.2015, p. 715-722.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Validation of eGFP fluorescence intensity for testing in vitro cytotoxicity according to ISO 10993-5
AU - Miller, Felicitas
AU - Hinze, Ulf
AU - Chichkov, Boris
AU - Leibold, Wolfgang
AU - Lenarz, Thomas
AU - Paasche, Gerrit
PY - 2015/12/24
Y1 - 2015/12/24
N2 - 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.
AB - 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.
KW - eGFP fluorescence
KW - implant material
KW - in vitro cytotoxicity test
KW - ISO 10993-5
UR - http://www.scopus.com/inward/record.url?scp=84983126777&partnerID=8YFLogxK
U2 - 10.1002/jbm.b.33602
DO - 10.1002/jbm.b.33602
M3 - Article
C2 - 26699416
AN - SCOPUS:84983126777
VL - 105
SP - 715
EP - 722
JO - Journal of Biomedical Materials Research - Part B Applied Biomaterials
JF - Journal of Biomedical Materials Research - Part B Applied Biomaterials
SN - 1552-4973
IS - 4
ER -