Density-controlled 3D bioprinting approach for fabrication of hierarchical tissue substitutes

This thesis presents the development of an advanced integrated 3D bioprinter system, incorporating an innovative optical particle/cell counting setup, with the objective of enhancing precision in fabricating cell-laden scaffolds for tissue engineering. The research addresses a critical challenge in the field: achieving precise control over cell distribution within bioprinted structures, a key determinant of functionality in tissue engineering and regenerative medicine (TERM). Central to the project is the design and construction of a real-time optical cell counter, utilizing laser light scattering to accurately monitor and regulate cell density during the bioprinting process. By ensuring precise cell concentrations throughout the printed scaffolds, this system contributes to the successful development of engineered tissues with reliable cellular architecture. The optical setup is designed with a 635 nm laser, which is directed through a series of mirrors and lenses into a microfluidic channel containing the cell suspension. As cells traverse the measurement zone, their interaction with the laser generates scattered light that is captured by a photodetector. This scattering data is immediately processed to determine both the number and size of cells in real time. This real-time feedback enables dynamic adjustments to be made during the printing process, ensuring consistent and accurate cell distribution across the entire structure. This novel approach effectively addresses a primary limitation in bioprinting—the challenge of maintaining uniform cell density across complex, printed tissues. The 3D bioprinting system was validated through a series of experiments, designed to test its ability to handle different concentration profiles. These experiments began with the identification of suitable structural designs for printing, where filled square configurations were found to offer mechanical stability and sufficient printing duration. This allowed for the application of various concentration profiles throughout the printing process. Confocal microscopy was employed to assess the final distribution of cells within the printed structures, confirming the ability of this new system to preserve the targeted cell concentration across the entire scaffold depth. A key highlight of the research is the successful implementation of gradient concentration profiles, mimicking the natural cellular gradients that are fundamental in biological tissues. The optical cell counter played a crucial role in ensuring seamless transitions between high and low concentration zones within these gradient-printed structures. This capability not only demonstrates the precision of this platform but also showcases the adaptability in generating sophisticated tissue constructs with complex cell density patterns. Ultimately, the results of this research underscore the bioprinter’s significant potential for advancing the field of tissue engineering. By enabling precise control over cellular architectures within bioprinted structures, this system paves the way for more accurate and reliable production of engineered tissues, offering a valuable tool for future applications in regenerative medicine and other biomedical fields.