Freestanding 3-D microvascular networks made of alginate hydrogel as a universal tool to create microchannels inside hydrogels

The diffusion of molecules such as nutrients and oxygen through densely packed cells is impeded by blockage and consumption by cells, resulting in a limited depth of penetration. This has been a major hurdle to a bulk (3-D) culture. Great efforts have been made to develop methods for generating branched microchannels inside hydrogels to support mass exchange inside a bulk culture. These previous attempts faced a common obstacle: Researchers tried to fabricate microchannels with gels already loaded with cells, but the fabrication procedures are often harmful to the embedded cells. Herein, we present a universal strategy to create microchannels in different types of hydrogels, which effectively avoids cell damage. This strategy is based on a freestanding alginate 3-D microvascular network prepared by in-situ generation of copper ions from a sacrificial copper template. This alginate network could be used as implants to create microchannels inside different types of hydrogels. This approach effectively addresses the issue of cell damage during microfabrication and made it possible to create microchannels inside different types of gels. The microvascular network produced with this method is (1) strong enough to allow handling, (2) biocompatible to allow cell culturing, and (3) appropriately permeable to allow diffusion of small molecules, while sufficiently dense to prevent blocking of channels when embedded in different types of gels. In addition, composite microtubules could be prepared by simply pre-loading other materials, e.g., particles and large biomolecules, in the hydrogel. Compared with other potential strategies to fabricate freestanding gel channel networks, our method is more rapid, low-cost and scalable due to parallel processing using an industrially massproducible template. We demonstrated the use of such vascular networks in creating microchannels in different hydrogels and composite gels, as well as with a cell culture in a nutrition gradient based on microfluidic diffusion. In this way, the freestanding hydrogel vascular network we produced is a universal functional unit that can be embedded in different types of hydrogel; users will be able to adopt this strategy to achieve vascular mass exchange in the bulk culture without changing their current protocol. The method is readily implementable to applications in vascular tissue regeneration, drug discovery, 3-D culture, etc.