Quantification of collagen matrix deposition in 2D cell cultures: a comparative study of existing assays

Collagen matrix deposition is an important biomarker to predict the regenerative capacity of new biomaterials or the therapeutic potential of new drugs in collagen-associated diseases. Several methods for the quantification of matrix collagen in tissue samples are established, e.g., Picro-Sirius red assay, hydroxyproline assay, antibody-based assays, or the 3,4-DHPAA-based assay. These methods have been extended to quantify deposited collagen in in vitro cell culture models, although their applicability has been questioned due to the much lower concentration and eventually lower relative abundance of deposited collagen in cell cultures than in tissue. Here we compare the performance of the above-mentioned methods for the quantification of deposited matrix collagen in 2D cell cultures under different conditions: culture time, addition of collagen deposition-stimulating molecules, and post-culture processing step (decellularization). We show that the available methods can deliver accurate results within different experimental windows. We provide a comprehensive analysis of the relevant experimental parameters that influence the assay, and the sensitivity limits for the different methods, as well as the involved effort. In a comparative table, we provide guidance for the selection of the most appropriate collagen quantification assay for different culture conditions.