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 post-printing 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.