Despite the growing relevance of 3D cell culture as a research model, imaging techniques used to characterize these models are highly limited. Due to the thickness and opacity of 3D cellular structures, most current imaging technologies cannot penetrate to the center of the tissues, resulting in only the outer 2 to 3 layers of cells being detected. This causes the dark centers often seen in images of 3D cell culture models, which proves highly problematic for accurate analysis as these outer cells are most exposed to compounds, nutrients and oxygen and thus do not reflect the entire cell population.

Dulbecco’s Modified Eagle Medium (DMEM) is modified to contain four times the amino acid and vitamin concentration of original Eagle’s Minimal Essential Medium. The original DMEM formulation contained a low concentration of glucose (1 g/L) and sodium pyruvate. However, DMEM is now available in a variety of formulations, including those with higher glucose levels and with or without sodium pyruvate. DMEM also uses a sodium bicarbonate buffer system, allowing for maintenance of physiological pH in a 5–10% CO2 environment. Importantly, DMEM commonly requires fetal bovine serum (FBS) supplementation.