Validation of in vitro assays in three-dimensional human dermal constructs

Area of Science:

• Biomaterial Science
• Tissue Engineering
• Cell Biology

Background:

• Three-dimensional (3D) cell culture systems are essential for accurate biomaterial cytocompatibility testing.
• Existing in vitro models often lack the complexity to fully represent in vivo conditions.

Purpose of the Study:

• To develop and optimize 3D in vitro dermal skin models for evaluating biomaterial cytocompatibility.
• To identify the most suitable cell viability assay for these 3D constructs.

Main Methods:

• Development of murine and human 3D dermal constructs using fibroblasts within collagen matrices.
• Evaluation of CellTiter-Blue, RealTime-Glo MT, and CellTiter-Glo viability assays.
• Z-stack imaging for cell morphology and distribution analysis.

Main Results:

• CellTiter-Glo was identified as the optimal assay for 3D dermal constructs, outperforming CellTiter-Blue and RealTime-Glo.
• CellTiter-Blue altered normal human dermal fibroblast morphology, impacting viability data reliability.
• RealTime-Glo was unsuitable due to linearity issues with cell density.
• Optimized shaking time for CellTiter-Glo enhanced reagent penetration and ATP release, revealing significant cell viability within the matrix.

Conclusions:

• The developed 3D human dermal construct provides a robust platform for biomaterial cytocompatibility assessment.
• CellTiter-Glo assay, with optimized parameters, reliably quantifies cell viability in 3D dermal models.
• Further development could lead to full-thickness skin models for enhanced testing capabilities.