Cell viability assessment using the Alamar blue assay: a comparison of 2D and 3D cell culture models

Comparisons of 2D and 3D cell culture models in literature have indicated differences in cellular morphology and metabolism, commonly attributed the better representation of in vivo conditions of the latter cell culture environment. Thus, interest in the use of 3D collagen gels for in vitro analysis has been growing. Although comparative studies to date have indicated an enhanced resistance of cells on collagen matrices against different toxicants, in the present study it is demonstrated that non-adapted protocols can lead to misinterpretation of results obtained from classical colorimetric dye-based cytotoxic assays. Using the well established Alamar blue assay, the study demonstrates how the transfer from 2D substrates to 3D collagen matrices can affect the uptake of the resazurin itself, affecting the outcome of the assay. Using flow cytometry, it is demonstrated that the cell viability is unaffected when cells are grown on collagen matrices, thus the difference seen in the fluorescence is a result of a dilution of the resazurin dye in the collagen matrix, and an increased uptake rate due to the larger cell surface exposed to the surrounding environment, facilitating more effective diffusion through the cellular membrane. The results are supported by a rate equation based simulation, verifying that differing uptake kinetics can result in apparently different cell viability. Finally, this work highlights the feasibility to apply classical dye-based assays on collagen based 3D cell culture models. However, the diffusion and bioavailability of test substances in 3D matrices used in in vitro toxicological assays must be considered and adaption of the protocols is necessary for direct comparison with the traditional 2D models. Moreover, the observations made based on the resazurin dye can be applied to drugs or nanoparticles which freely diffuse through the collagen matrices, thus affecting the effective concentration exposed to the cells.