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Microfluidic systems represent a key-enabling platform for novel diagnostic tools for use at the point-of-care in clinical contexts as well as for evolving single cell diagnostics. The design of 3D microfluidic systems is an active field of development, but construction of true interconnected 3D microfluidic networks is still a challenge, in particular when the goal is rapid prototyping, accurate design and flexibility. We report a novel approach for the construction of programmable 3D microfluidic systems consisting of modular 3D template casting of interconnected threads to allow user-programmable flow paths and examine its structural characteristics and its modular function. To overcome problems with thread template casting reported in the literature, low-surface-energy polymer threads were used, that allow solvent-free production. Connected circular channels with excellent roundness and low diameter variability were created. Variable channel termination allowed programming a flow path on-the-fly, thus rendering the resulting 3D microfluidic systems highly customizable even after production. Thus, construction of programmable/reprogrammable fully 3D microfluidic systems by template casting of a network of interconnecting threads is feasible, leads to high-quality and highly reproducible, complex 3D geometries. [ABSTRACT FROM AUTHOR]