Clonogenic Survival Analysis of 3D Bioprinted Irradiated Tumor Constructs
Abstract
Purpose
A novel method for assessing clonogenic survival in irradiated 3D bioprinted tumor models.
Methods
A549 cells were suspended in a 2% (w/v) alginate and 3% (w/v) gelatin hydrogel at a density of ~3 x 106 cells/mL and used 3D extrusion-based bioprinting to construct solid discs (radius 5 mm, height 0.3 mm). The constructs were crosslinked and incubated for 14 days after which they were irradiated to doses of 0, 2, 4, and 8 Gy. Twenty-four hours post-irradiation, the constructs were decrosslinked and seeded at dose-dependent cell densities for the clonogenic survival assay. After 10-14 days of incubation, colonies were fixed and stained with crystal violet. Colony counts were quantified using ImageJ with an image-processing protocol. Plating efficiency was calculated from the control samples and used to compute the surviving fraction.
Results
Control wells exhibited a plating efficiency of ~14%. Clonogenic survival decreased with increasing radiation dose. The mean surviving fraction computed over three samples was 0.27 ± 0.05, 0.022 ± 0.007, and 0.0018 ± 0.0008 at 2, 4, and 8 Gy, respectively. The survival curve demonstrated a nonlinear, dose-dependent reduction in survival.
Conclusion
These findings support the utility of a method for assessing clonogenic survival in extrusion-based 3D bioprinted cancer constructs following irradiation.