We describe micromanipulation and microinjection procedures for the fabrication of soft-matter networks consisting of lipid bilayer nanotubes and surface-immobilized vesicles. These biomimetic membrane systems feature unique structural flexibility and expandability and, unlike solid-state microfluidic and nanofluidic devices prepared by top-down fabrication, they allow network designs with dynamic control over individual containers and interconnecting conduits. The fabrication is founded on self-assembly of phospholipid molecules, followed by micromanipulation operations, such as membrane electroporation and microinjection, to effect shape transformations of the membrane and create a series of interconnected compartments. Size and geometry of the network can be chosen according to its desired function. Membrane composition is controlled mainly during the self-assembly step, whereas the interior contents of individual containers is defined through a sequence of microneedle injections. Networks cannot be fabricated with other currently available methods of giant unilamellar vesicle preparation (large unilamellar vesicle fusion or electroformation). Described in detail are also three transport modes, which are suitable for moving water-soluble or membrane-bound small molecules, polymers, DNA, proteins and nanoparticles within the networks. The fabrication protocol requires ∼90 min, provided all necessary preparations are made in advance. The transport studies require an additional 60-120 min, depending on the transport regime. © 2011 Nature America, Inc. All rights reserved.