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K. Birke, N. Kopsachilis, T. Lindl, M. T. Birke, F. E. Kruse, U. C. Welge-Luessen; Evaluation of the Applicability of the MTT Viability Assay in 3-Dimensional Hemi-Cornea Constructs. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6237.
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To test the MTT viability assay for cytotoxic site effects in 3-dimensional hemi-cornea constructs compared to the CCK-8 assay.
3-dimensional hemi-cornea constructs were produced with SV40 transformed human corneal keratinocytes and SV40 transformed human corneal epithelial cells in an air-liquid system. Viability measurements were done using MTT or CCK-8 assay, respectively. Histological analyses were done on semi- and ultra-thin sections of constructs by light and electron microscopy. Formazan salt formation was visualized by metal salt staining according to Gomori. The level of cell death in the constructs was assessed by TUNEL staining. TGF-β1 staining was used as a marker for stress induction in keratinocytes.
The setup of 3-dimensional hemi-cornea constructs was highly reproducible and the constructs were comparable to human corneas. Viability measurements on 20 analogously produced constructs by MTT assay did not provide reproducible results, whereas CCK-8 assays provided reproducible measurements. By histological analyses, a significant level of cell degradation was observed in the constructs after incubation with MTT. On the electron microscopic level increased numbers of necrotic cells were observed in all layers of the constructs. CCK-8 incubation, in contrast, did not induce increased cell degradations. By metal salt stainings according to Gomori, additional formation of formazan salt was detected in deeper layers of the constructs than detected by MTT stainings. By TUNEL stainings, a significant increase of cell death was detected in MTT incubated constructs compared to CCK-8 incubated constructs. Moreover, already 30 minutes after MTT incubation, keratinoytes expressed TGFβ1, which was not observed in CCK-8 incubated constructs.
MTT assay data were not reproducible and did not agree with the histological results. Moreover, the MTT assay induced cell degeneration even in deeper layers of the constructs. The CCK-8 assay, in contrast, had no cytotoxic side effects and provided highly reproducible data. Therefore, the MTT assay is not the method of choice for viability measurements in 3-dimensional hemi-cornea constructs.
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