A novel integrated technology chain of laser-microstructured multilayer foils for fast, flexible, and low-cost manufacturing of lab-on-a-chip devices especially for complex cell and tissue culture applications, which provides pulsatile fluid flow within physiological ranges at low media-to-cells ratio, was developed and established. Initially the microfluidic system is constructively divided into individual layers, which are formed by separate foils or plates. Based on the functional boundary conditions and the necessary properties of each layer, their corresponding foils and plates are chosen. In the third step, the foils and plates are laser microstructured and functionalized from both sides. In the fourth and last manufacturing step, the multiple plates and foils are joined using different bonding techniques like adhesive bonding, welding, etc. This multilayer technology together with pneumatically driven micropumps and valves permits the manufacturing of fluidic structures and perfusion systems, which spread out above multiple planes. Based on the established labon-a-chip platform for perfused cell-based assays, a multilayer microfluidic system with two parallel connected cell culture chambers was successfully implemented.