Abstract
Tissue engineering in combination with stem cell technology has the potential to revolutionize human healthcare. It aims at the generation of artificial tissues that can mimic the original with complex functions for medical applications. However, even the best current designs are limited in size, if the transport of nutrients and oxygen to the cells and the removal of cellular metabolites waste is mainly dependent on passive diffusion. Incorporation of functional biomimetic vasculature within tissue engineered constructs can overcome this shortcoming. Here, we developed a novel strategy using 3D printing and injection molding technology to customize multilayer hydrogel constructs with pre-vascularized structures in transparent Polydimethysiloxane (PDMS) bioreactors. These bioreactors can be directly connected to continuous perfusion systems without complicated construct assembling. Mimicking natural layer-structures of vascular walls, multilayer vessel constructs were fabricated with cell-laden fibrin and collagen gels, respectively. The multilayer design allows functional organization of multiple cell types, i.e., mesenchymal stem cells (MSCs) in outer layer, human umbilical vein endothelial cells (HUVECs) the inner layer and smooth muscle cells in between MSCs and HUVECs layers. Multiplex layers with different cell types showed clear boundaries and growth along the hydrogel layers. This work demonstrates a rapid, cost-effective, and practical method to fabricate customized 3D-multilayer vascular models. It allows precise design of parameters like length, thickness, diameter of lumens and the whole vessel constructs resembling the natural tissue in detail without the need of sophisticated skills or equipment. The ready-to-use bioreactor with hydrogel constructs could be used for biomedical applications including pre-vascularization for transplantable engineered tissue or studies of vascular biology.
Highlights
The aim of tissue engineering is to generate artificial tissues or organs with complex functions for replacement therapy, disease modeling and drug screening through a combination of desired cell types, bioactive molecules and biomaterials (Biondi et al, 2008; Khademhosseini et al, 2009; Atala et al, 2012; Abaci et al, 2016)
Our results suggest that (1) multicellular hydrogel constructs with vesselmimicking structure can be fabricated in situ within customized PDMS bioreactors and (2) the constructs in this system can maintain multilayer structure under continuous perfusion in culture
We applied widely available low-cost 3D printing technology in conjugation with soft lithography and molding to develop ready-to-use multilayered, multicellular tissues with embedded vasculature inside custom PDMS bioreactors
Summary
In various types of engineered tissue constructs, cell adhesion, proliferation, differentiation as well as extracellular matrix production has been studied (ECM; Agarwal et al, 2017; Phan et al, 2017). Most of these constructs share a size limitation rooted in the metabolic needs of the embedded cells (Novosel et al, 2011). Cell survival is usually restricted within a limited range of 150–200 μm from the nearest capillary In this area, the cells receive sufficient supply including oxygen, nutrition, and growth factors (Kannan et al, 2005). For the generation of functional tissues in clinically relevant sizes, inclusion of a functional volumetric and biomimetic vasculature is mandatory
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