Skeletal muscle is a complex tissue composed of multinucleated myo bres responsible for force generation, supported by multiple cell types. Many severe and lethal disorders affect skeletal muscle; therefore, engineering models to reproduce such cellular complexity and function is instrumental for investigating muscle pathophysiology and developing therapies. Here, we detail the modular 3D bioengineering of multilineage skeletal muscles from human induced pluripotent stem cells, which are rst differentiated into myogenic, neural and vascular progenitor cells, and then combined within 3D hydrogels under tension to generate an aligned myo bre scaffold containing vascular networks and motor neurons. 3D bioengineered muscles recapitulate morphological and functional features of human skeletal muscle, including establishment of a pool of cells expressing muscle stem cell markers.Importantly, bioengineered muscles provide a high-delity platform to study muscle pathology, such as emergence of dysmorphic nuclei in muscular dystrophies caused by mutant lamins. The protocol is easy to follow for operators with cell culture experience and takes between 9 and 30 days, depending on the number of cell lineages in the construct. We also provide examples of applications of this advanced platform for testing gene and cell therapies in vitro, as well as for in vivo studies, providing proof-ofprinciple of its potential as a tool to develop next-generation neuromuscular or musculoskeletal therapies.

Commercially available cell culture devices are designed to increase the complexity of simple cell culture models to provide better experimental platforms for biological systems. From microtopography, microwells, plating devices and microfluidic systems to larger constructs for specific applications like live imaging chamber slides, a wide variety of culture devices with different geometries have become indispensable in biology labs. However, the techniques used for their fabrication can be out of reach for most wet labs due to cost and availability of specialised equipment or the need for engineering expertise. Moreover, these techniques also have technical limitations to the volumes, shapes and dimensions they can generate. For these reasons, creating customisable devices tailored to lab-specific biological questions remains difficult to apply. Taking advantage of low-cost, high-resolution desktop resin 3D printers combined with PDMS soft-lithography we have developed an optimised microfabrication pipeline capable of generating a wide variety of customisable devices for cell culture and tissue engineering in an easy, fast reproducible way for a fraction of the cost of conventional microfabrication or commercial alternatives. This technique enables the manufacture of complex devices across scales bridging the gap between microfabrication and fused deposition moulding (FDM) printing. The method we describe allows for the efficient treatment of resin-based 3D printed constructs for PDMS curing, using a combination of curing steps, washes and surface treatments. Together with the extensive characterisation of the fabrication pipeline, we provide several proof-of-principle applications ranging from simple 2D culture devices to large tissue engineering constructs and organoid formation systems. We believe this methodology will be applicable in any wet lab, irrespective of prior expertise or resource availability and will therefore enable a wide adoption of tailored microfabricated devices across many fields of biology.