Organ-on-chip (OoC) devices are increasingly used for biomedical research and to speed up the process of bringing a medicinal drug from the lab to the market. The technology also addresses ethical issues linked to animal testing as it offers a reduction in animal experiments through its possibility to mimic the environment in the human body. Hence, it produces more relevant results than plain cell-culture experiments, while still sharing the possibility of high-throughput testing by simultaneously operating many devices in parallel. The main components of an OoC device are microfluidic channels and porous membranes. Current chips are often assembled from several parts. In the development phase a small change in design will cause a delay in the research because a new prototype has to be built again step-by-step. This research addresses this problem by targeting the fabrication of a microfluidic device in a single lithography and development step. A device consisting of two crossed channels at different heights separated by a membrane was chosen. The manufacturing method used was two-photon lithography (TPL) on positive photoresist AZ 4562. TPL exploits the fact that two-photon absorption non-linearly depends on the light intensity. The required intensity is only achieved in a small volume around the focal point, called the voxel. By positioning and moving this voxel inside the resist, 3D manufacturing is possible. Therefore the technique can be used for the single step fabrication of a closed channel. By vertically moving the voxel through the resist, pores can be manufactured. In literature an ellipsoidal voxel shape is assumed, leading to the assumption that the maximum voxel, and therefore pore diameter is found at the focal plane, i.e. at z = 0. However, this research shows that the voxel may also have an hourglass-shape for a high enough laser intensity. In this case the maximum voxel diameter is found at a distance z from the focal plane. For the production of pores, therefore the maximum voxel diameter instead of the diameter at the focal plane has to be taken into account. The smallest pores produced measured 250 nm - 290 nm. AZ 4562 was used as a stamp and a mold for the manufacturing of 3D topographical features on a PDMS surface. Finally the microfluidic device was successfully produced in a layer of 50 µm thick resist. The channel width was 100 µm and the height was 10 µm. The channels were separated by 10 µm in height. The sizes of the input and output holes were adapted to the diameter of the smallest available blunt needle, which is 210 µm.