Printing-induced cell injury evaluation during laser printing of 3T3 mouse fibroblasts

Three-dimensional bioprinting has emerged as a promising solution for the freeform fabrication of living cellular constructs, which can be used for tissue/organ transplantation and tissue models. During bioprinting, some living cells are unavoidably injured and may become necrotic or apoptotic cells. This study aims to investigate the printing-induced cell injury and evaluates injury types of post-printing cells using the annexin V/7-aminoactinomycin D and FAM-DEVD-FMK/propidium iodide assays during laser printing of NIH 3T3 mouse fibroblasts. As observed, the percentage of post-printing early apoptotic mouse fibroblasts increases with the incubation time, indicating that postprinting apoptotic mouse fibroblasts have different initiation lag times of apoptosis due to different levels of mechanical stress exerted during laser printing. Post-printing necrotic mouse fibroblasts can be detected immediately after printing, while post-printing early apoptotic mouse fibroblasts need time to develop into a late apoptotic stage. The minimum time needed for post-printing early apoptotic mouse fibroblasts to complete their apoptosis pathway and transition into late apoptotic mouse fibroblasts is from 4 h to 5 h post-printing. The resulting knowledge of the evolution of different apoptotic post-printing mouse fibroblasts will help better design future experiments to quantitatively determine, model, and mitigate the post-printing cell injury based on molecular signal pathway modeling.