Fig. 1

Analysis of cell viability in 2D and 3D cultures. a Comparison of viability and metabolic activity assessment via spectrophotometric readouts or (confocal) microscopy in 2D and 3D in vitro culture setups. b 2D cultures are shown as monolayers in well plates or tissue culture flasks where reagents have direct contact with the cells. c 3D samples are characterized by the presence of a biomaterial as a supportive structure (e.g., fabricated via 3D bioprinting) in which the cells are embedded. Reagents need to diffuse through the material to stain or be metabolized by the cells, and thus, a limitation in penetration depth may result in low or no positive staining toward the center of the 3D construct, as illustrated for live-dead staining. Furthermore, many biomaterials or compounds present in the materials can interfere and show an autofluorescent signal that, in the worst case, results in higher readout values or background staining than that derived from the cells only