No abstract

Stem cell‐based experimental platforms for neuroscience can effectively model key mechanistic aspects of human development and disease. However, conventional culture systems often overlook the engineering constraints that cells face in vivo. This is particularly relevant for neurons covering long range connections such as spinal motor neurons (MNs). Their axons extend up to 1m in length and require a complex interplay of mechanisms to maintain cellular homeostasis. However, shorter axons in conventional cultures may not faithfully capture important aspects of their longer counterparts. Here this issue is directly addressed by establishing a bioengineered platform to assemble arrays of human axons ranging from micrometers to centimeters, which allows systematic investigation of the effects of length on human axonas for the first time. This approach reveales a link between length and metabolism in human MNs in vitro, where axons above a “threshold” size induce specific molecular adaptations in cytoskeleton composition, functional properties, local translation, and mitochondrial homeostasis. The findings specifically demonstrate the existence of a length‐dependent mechanism that switches homeostatic processes within human MNs. The findings have critical implications for in vitro modeling of several neurodegenerative disorders and reinforce the importance of modeling cell shape and biophysical constraints with fidelity and precision in vitro.

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.