3D Bioprinting of Bladder Tumor Organoids for Anti-Cancer Drug Screening

Abstract

Bioprinting techniques have long been considered to provide the next-generation anti-cancer drug screening platform attributing to their capability to pattern the cells and ECMs with the aim of recapitulating the disease conditions. For this purpose, several models have been developed with the tumor constituting cellular and ECM components, such as the tumor cells, stromal cells, immune cells, and the natural ECMs. It was demonstrated that the tumor cells are more susceptible to the drugs when the additional components are absent. However, such models have several limitations considering the biological relevance and engineering rationale. First, the bioinks have been poorly designed, and they are incapable of fully recapitulating the tumor tissues. Cell lines are used mainly because of their easy acquisition and handling. The biomaterials are not carefully chosen for mimicking the tumor microenvironment, being dependent on the ion- and UV-crosslinking hydrogels. They might be suitable for showing the feasibility of bioprinting technique to print the cells, but they are functionally depart from the tumor microenvironment. Moreover, the consequent constructs are too large either to produce large number of constructs and to reproduce the identical models. It decreases not only the reliability of this method but also the applicability. Lastly, the importance of tumor heterogeneity has been largely neglected, and the current models are focused on co-culturing the tumor cells and the stromal components. The aims of this thesis are to improve the biological relevance of the current models and to fully elicit the benefit of the bioprinting technique. In order to generate the tumor models with only the biologically relevant sources, the cellular and stromal components were derived from the patients, and they were suspended in the natural ECMs. They were initially printed in an array of 10  l droplets, and treated against the chemotherapeutic agent, cisplatin. To observe the influence of the stromal components in inducing the drug resistance, two groups of tumor models were generated, with one group being supplemented with stromal components, including the cancer-associated fibroblasts and the endothelial cells. The stroma supplemented group showed high resistance against the drugs, which was in accordance with the manually generated tumor models. Although this model satisfied the biological requirements, they were limited to the simple mixture of cells and hydrogels because the natural ECMs were nearly impossible to be patterned with conventional bioprinting methods. Therefore, a new strategy was devised to enable 3D patterning, termed as core-shell printing. Contrary to the layer-by-layer deposition methods, the core-shell printing method performs the layer deposition process at the core of the constructs. As the bioink of the core layer pushes the external layers outwards, it leads to the formation of another sphere at the center of the constructs. The printing parameters and the material properties were optimized for the stability and the reproducibility of the process. This technique was applicable to various types of hydrogels regardless of their crosslinking properties, and was able to generate up to triple layers with only 50  l of the bioink. The consequent constructs were as small as 5 mm, and the internal structure was maintained after long-term culture and the contraction. The core-shell printing method was further applied to generate the bladder tumor models. Cancer-associated fibroblasts were printed at different densities, in which the drug resistance significantly increased in the models with higher cell densities. Despite the improvements, the above models were dependent on using the large number of tumor organoids. In order to observe the tumor heterogeneity, the individual tumor cells need to be separated for further analysis. Since the microextrusion technique deposits the large number of cells continuously, it has been unable to deliver the cells on the single-cell level. In this respect, the piezoelectric inkjet printing method was used with their capability to eject a tiny amount of droplets. The tumor organoids were enzymatically dissociated and suspended in the culture medium to be used as the bioink. They were separately delivered into ultralow adhesion treated microwell plates, and cultured into the tumor organoids. The printing parameters were optimized to maximize the efficiency, so as the culture medium condition for their survivial. The single cell-derived tumor organoids were highly heterogeneous regarding the morphology, mRNA expression, protein expression, and the drug responses. Through the research, the reliability of the bioprinting technique in generating the drug test model has been significantly improved concerning the biological and structural relevance, so as the applicability of this method for observing the tumor heterogeneity. This thesis paves the path of the engineering techniques for their translation towards the biomedical application.