Establishment of three-dimensional tumor microenvironment in an In-Vitro coculture system using lung cancer stem cells through a three-dimensional bioprintingmethod

Objective: Three-dimensional (3D) bioprinting technology, one of the most widely used methods in tissue engineering and regenerative medicine, is an important technique used to elucidate many physiological mechanisms, including stem cell studies and drug research. Despite the advances in this technology, complete non-small cell lung cancer (A549) models are insufficient to mimic real lung tumor tissue. This study, aimed to 3D bioprint and characterize lung cancer stem cells, human endothelial cells (EA.hy926), and healthy lung epithelial cells (BEAS-2B) by the tumor microarchitecture and air-liquid interface to create a morphological and functional 3D organoid-type bioprinting method for in vivo tumor microenvironment. Method: EA.hy926 cells were seeded and plated on a transwell membrane by the in-vivo microarchitecture scheme. BEAS-2B and CCS cells were cultured at the air-liquid interface by 3D bioprinting using two types of bioinks we developed by their properties. The resulting lung 3D tumor microenvironment organoid was characterized by live/dead, SEM, histochemistry, and immunofluorescence analysis. Results: Both bioink 1 (BEAS-2B cells) and bioink 2 (BEAS-2B and CCS) were consistent with in vivo microarchitecture and cell-specific markers in terms of cell viability, pore diameter, and cell size. Conclusion: These developed bioinks, bioprinting design, and air-liquid interface models need further studies using bioreactors that allow for long-term culture and the addition of other cells in the tumor microenvironment.