One of the main challenges in tissue engineering today is to create a complete network of blood vessels and capillaries throughout an artificial tissue. A promising solution, now demonstrated by researchers in Belgium, is to fabricate micron-sized cellular building blocks that already incorporate a capillary network, which can then be 3D bioprinted to form a large tissue structure. As well as helping to engineer large-area tissues or organs, the technique would also be useful for making in vitro structures that can be used in cancer research, drug testing and disease modelling.
Tissue engineered constructs are useful in many applications, including as in vitro models for injury, disease, drug-screening, or to repair, regenerate or replace dysfunctional tissues or organs. Much progress is being made in the field, but it is easier to engineer thin tissues with a low metabolism, such as skin or cartilage, than to make thick 3D tissues. The problem is that cells cannot diffuse more than around 100 to 200 µm in space, which means that those lying deep in the core of a large engineered construct have insufficient access to nutrients and oxygen and are unable to survive.
In living organisms, this nutrient- and oxygen exchange between blood and tissues occurs in blood vessels and capillaries, collectively known as the vasculature. “We therefore need to find a way to make such a structure in engineered 3D tissue – and make sure that it extends throughout the entire construct,” explains Heidi Declercq of Ghent University, who led this study. “A complete vascular tree, ranging in size from millimetres to microns, is required, since nutrient- and oxygen-exchange occurs mainly in the microvasculature.”
Although larger vessels can be fabricated by incorporating printed channels composed of bioinks – biomaterials laden with cells that can be printed to form tissue – smaller vasculature networks are more difficult to make in this way because of the limited resolution of bioprinting techniques.
Cellular building blocks
Declercq and colleagues instead turned to cellular self-assembly, a bottom-up approach for building large tissue constructs. “We use spheroids or microtissues with a specific microarchitecture as building blocks,” Declercq explains. “Small and uniform-shaped spheroids are made spontaneously by seeding cells on microwells, which are created using a polymer mould containing 2865 pores with a diameter of 200 microns.”
When a cell suspension is seeded onto the microwells, gravity causes the cells to fall to the bottom of the pores. Here they are forced to interact with other, which causes the cells to self-assemble into spheroids.
“The properties of the spheroids produced in this way depend on the cells they contain and the cell types used to ‘support’ them,” continues Declercq. “Endothelial cells, like the ones studied in this work, can be co-cultured with supporting fibroblasts or mesenchymal stem cells to promote angiogenesis [the formation of blood vessels].”
These spheroids can then be directly assembled by 3D bioprinting to form a macroscale tissue structure. “This strategy is based on cell sorting and microtissue fusion,” says Declercq. “Cells organized into a spheroid can fuse into a macrotissue in a process that can be explained by the ‘differential adhesion hypothesis’. This says that multicellular tissues behave like liquids, thanks to their surface tension, and will rearrange and merge to maximize their adhesive bonds and minimize their free energy.”
Spheroids enable bioprinting
By seeding 750 000 cells onto one microwell, the researchers produced 2865 spheroids containing roughly 262 cells/spheroid. These spheroids measure around 125 µm across, a size that is compatible with bioprinting techniques that employ needles with diameters in the 200 µm range.
“In our study, we co-cultured human umbilical-vein endothelial cells (HUVECs) with human foreskin fibroblasts (HFF) and adipose-tissue-derived mesenchymal stem cells (ADSCs) in different ratios,” says Declercq. “We tested different compositions and found that a 1:9 ratio of HUVEC/supporting-cells produced the most stable spheroids.”
The researchers found that capillary-like networks formed in spheroids that included ADSCs, with larger diameter spheroids (>170 µm) forming a more branched capillary-like structure. They also showed that individual spheroids in suspension fuse together within just 24 hours, and within 4 days a branched capillary-like network extends throughout the entire construct. Even when embedded in a hydrogel – which would be needed to create a bioink – spheroids started to fuse together within 18 hours.
The researchers, reporting their work in the IOP journal Biofabrication, say that they will now undertake in vitro experiments to help them select the best bioinks for making their vascularized constructs. “We will also set up in vivo experiments to see how the constructs connect with real host tissue,” adds Declercq.
- Read our special collection “Frontiers in biofabrication” to learn more about the latest advances in tissue engineering. This article is one of a series of reports highlighting high-impact research published in Biofabrication.