Development and Characterization of a 3D-Bioprinted Lung Tumor Model for Preclinical Radiotherapy Studies
Abstract
Purpose
To develop and characterize a 3D-bioprinted non-small cell lung cancer (NSCLC) model as a reproducible and physiologically relevant platform for pre-clinical radiotherapy studies, with initial assessment of cell growth, spatial distribution, and radiation-response.
Methods
The metastatic human NSCLC cell line (A549) was mixed with an in-house 2% (w/v) alginate and 3% (w/v) gelatin hydrogel to form the bioink. Three-dimensional, 0.3 mm-thick S-shaped constructs were fabricated using extrusion-based bioprinting to promote oxygen and nutrient diffusion and printed directly into 24-well culture plates. Following ionic cross-linking with CaCl2, constructs were maintained in culture for up to 14 days prior to irradiation. To assess radiation response, bioprints were irradiated to 0, 2, or 6 Gy using the CellRad irradiator (100 kV, 5 mA). Cell viability was evaluated using LIVE/DEAD fluorescence staining and confocal microscopy (Leica Microsystems) with volume-based, z-stack imaging performed at 1, 3, and 7 days post- irradiation. Viability was assessed using the summed, normalized total live-cell signal area across z-slices (mean ± SD, n = 2), a metric proportional to viable cell number and less sensitive to spheroid-based counting noise.
Results
Viable A549 cells were observed to be uniformly distributed throughout the printed constructs, indicating sustained growth. Viability metrics demonstrated an inverse relationship between dose and live-cell area per construct, consistent with expected radiation dose-response behavior. No significant dependence on time post-irradiation was observed within the studied interval.
Conclusion
A 3D-bioprinted NSCLC model was successfully developed and shown to support cell growth, while exhibiting predictable radiation dose-response. This platform provides a controllable and repeatable 3D physiologically relevant model for radiobiology studies. Future work will evaluate how dose fractionation and spatial distribution influence endpoints such as immunogenic cell death and clonogenic survival, and will incorporate normal lung fibroblasts to assess pulmonary fibrosis.