Abstract 1316: Vegetal scaffold as radiobiology model to study radiation cancer response

Current in vitro tumor models have issues in accuracy in that the 2D structures and (often rare) cell co-culture technologies that exist, lack many features or characteristics found in vivo. The use of decellularized plant structures recellularized with human cells, aims to overcome these issues by taking advantage of their natural 3D structure. By using this approach on spinach leaves as a 3D scaffold, we have developed a new model that may be used as a new tumor model for radiobiology research. Spinach leaves were decellularized following serial chemical treatments with hexanes, SDS, Triton-X100 and bleach. In order to characterize the efficiency of the decellularization process, the rigidity of the leaves was assessed by Atomic Force Microscope (AFM) and DNA and protein quantification. Human prostate (PC3) and breast (MCF7) cancer cells were then seeded onto leaf. Seeding efficiency was assessed by optical microscopy and viability and proliferation ability were tested by MTT assay. In order to evaluate if the cells were biologically active, we then assessed radiation response. Extra cell-seeded leaves were irradiated and the expression of radiation-responsive genes were assessed in MCF7 cells. Additionally, DNA damage levels in PC3 cells were evaluated by γ-H2AX foci measurement using fluorescence microscopy. The decellularization process was successful, showing a protein content of 0.31 μg/mg tissue compared to the fresh leaf at 14.4 μg/mg tissue. The DNA quantity was similarly disparate between fresh and decellularized leaves. Microscopy showed that PC3 and MCF7 cells were well attached to the decellularized leaf surface after 24h incubation. Mechanical testing with AFM confirmed attachment by measuring Young's modulus values of 2.81, 88 and 197 MPa for decellularized, recellularized and fresh leaves respectively. Viability assays confirmed that cells were alive and able to proliferate. The gene expression assay showed changes in expression levels between 2D cell culture and cells seeded on leaves both at basal state and after 5Gy-irradiation in MCF7 cells. Finally, γ-H2AX immunofluorescent imaging showed DNA damage repairs are induced 1 hour after 5Gy of X-ray irradiation in PC3 cells and are effective up to 24h. Plant can be decellularized in order to create a 3D scaffold that may act as a support for cell seeding. Interestingly, radiation response can be measured on this new model and even show significant difference with standard 2D cell culture.