Methodological study of fibroblasts MEF 3T3 heterogeneity cultured on hydrogel substrates with different surface functionalization

Recently, cellular heterogeneity has gained attention in several scientific studies. It’s a phenomenon of the occurrence of differences in cellular parameters, for example, morphology or migration parameters of cells from a single cell line. It has been studied primarily in cancer cells; however, normal cells exhibit analogous behavior. This study investigated morphological and migratory parameters heterogeneity in mouse embryonic fibroblast (MEF 3T3) cells cultured on polyacrylamide (PA) substrates with elasticity of 20 and 40 kPa, which were functionalized with fibronectin solutions in 1, 5, 10 and 20 μg/ml concentrations. Two-hour time lapse microscopy made it possible to quantify the cells spread area and velocity. Based on these parameters and observations of cellular migration strategies, it was possible to classify normal cells into four subpopulations - mesenchymal, amoeboid, slow amoeboid and polygonal/bigonal. Cells grown on softer substrates (20 kPa) showed a greater sensitivity to fibronectin than those grown on stiffer substrates. Cells area was maximal, with the lowest velocity for cells grown on PA substrates functionalized with a 10 μg/ml solution, making their parameters closest to those grown on glass. Cells underwent spontaneous transitions between subpopulations, but no direct transition between mesenchymal and amoeboid or slow amoeboid has been observed. Overall, substrate elasticity and concentration of fibronectin in the functionalizing solution impact the morphology and migration parameters of cells. These findings demonstrate the occurrence of heterogeneity phenomenon in normal mouse fibroblasts. This study also shows the impact of the concentration of fibronectin in the functionalization solution on cells’ behavior.