Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

Abstract

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ-and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

Fabrication Techniques for Vascular and Vascularized Tissue Engineering

“…For example, 50 µm diameter microchannels were fabricated at a speed of 95 µm s −1 in a collagen type I hydrogel . Laser degradation allows for high resolution printing by confining the focal laser beam to an extremely small volume, meaning it is ideal for the generation of perfusable microvasculature . For example, 3D cerebral capillary networks were created in PEGDA hydrogels using laser degradation under the guidance of confocal microscope images (Figure B) .…”

Fundamentals of Laser‐Based Hydrogel Degradation and Applications in Cell and Tissue Engineering

“…Additionally, fabrication of well‐defined, repeatable vascular architectures lends itself to the development of fluidized disease models. In this respect, laser‐based degradation of hydrogels has facilitated the fabrication of in vitro biomimetic microfluidic networks for subsequent tissue vascularization …”

“…An image‐guided, laser‐based degradation technique has also been developed that allows for accurate recapitulation of the 3D architecture of microvascular networks in hydrogels . In this approach, any 3D configuration can be utilized from those generated using computer‐aided design (CAD) to 3D image stacks of in vivo vasculature.…”

“…In this regard, laser‐based degradation of hydrogels can be used to create “smart” microfluidic systems for material transport and diffusion‐based studies. Fluidic flow within fabricated microchannels can be tracked in real time by imaging fluorescent particles and/or small molecules as they flow through the microchannels and diffuse into the surrounding bulk hydrogel constructs ( Figure A–C) . Transport of fluorescent moieties from one microchannel to another adjacent microchannel can also be investigated in these systems (Figure A) .…”

“…Scale bar = 50 µm (white arrows indicate direction of flow in (c) and (d)). Reproduced with permission . Copyright 2017, JoVE.…”