Applying XTT, WST-1, and WST-8 to human chondrocytes: A comparison of membrane-impermeable tetrazolium salts in 2D and 3D cultures.

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  • Additional Information
    • Source:
      Publisher: Ios Press Country of Publication: Netherlands NLM ID: 9709206 Publication Model: Print Cited Medium: Internet ISSN: 1875-8622 (Electronic) Linking ISSN: 13860291 NLM ISO Abbreviation: Clin Hemorheol Microcirc Subsets: MEDLINE
    • Publication Information:
      Publication: Amsterdam : Ios Press
      Original Publication: Amsterdam ; Washington, DC : IOS Press, c1997-
    • Subject Terms:
      Cell Culture Techniques/*methods ; Chondrocytes/*metabolism ; Tetrazolium Salts/*metabolism; Humans
    • Abstract:
      Background: Tetrazolium-based assays are optimized to assess proliferation/toxicity of monolayer or suspension cells in microtiter plates. With regard to tissue engineering and regenerative medicine the need for in vivo like 3D microtissues has an increasing relevance. Applying tetrazolium-based assays to 3D culture systems is technically more challenging. The composed microenvironment may influence the assay standards, e.g. equal distribution of tetrazolium.
      Objective: Evaluation of membrane-impermeable tetrazolium salt-based assays with regard to spheroid culture (3D) of human chondrocytes.
      Methods: Chondrocytes were isolated from human articular cartilage. XTT, WST-1, and WST-8 were applied to monolayer cells (2D, varying cell numbers) and spheroids (3D, different sizes) in 96well plates. Formazan formation was measured spectrophotometrically after different incubation periods. Evaluation was done using phase contrast microsopy (toxicity), analyzing the correlation of cell number and absorbance signals (Gompertz function), and document signal over background ratio.
      Results: In monolayer culture the assays showed a correlation between seeded cell numbers and absorption data. Spheroid sizes are directly related to the starting cell number. A correlation between size and absorbance was only detectable starting from 10,000 cells/aggregate. Phase contrast microscopy of monolayer cells revealed strong toxicity effects of the WST-1 (4 h) and XTT (8 h) assay and no signs of toxicity using WST-8.
      Conclusion: The WST-8 assay is non-toxic and revealed the highest sensitivity in comparison to the XTT or WST-1 assay. There is evidence, that only cells of the outer rim of spheroids are able to convert membrane-impermeable tetrazolium salts to formazans.
    • Contributed Indexing:
      Keywords: 2D culture; 3D culture; WST-1; WST-8; XTT; human chondrocytes; spheroid
    • Accession Number:
      0 (Tetrazolium Salts)
    • Publication Date:
      Date Created: 20170905 Date Completed: 20180417 Latest Revision: 20181202
    • Publication Date:
      20221213
    • Accession Number:
      10.3233/CH-179213
    • Accession Number:
      28869462