On-chip high-definition bioprinting of microvascular structures

Agnes Dobos,Aleksandr Ovsianikov,Jasper Van Hoorick,Sandra Van Vlierberghe,Liesbeth Tytgat,Franziska Gantner,Marica Markovic

doi:10.1088/1758-5090/abb063

Abstract

‘Organ-on-chip’ devices which integrate three-dimensional (3D) cell culture techniques with microfluidic approaches have the capacity to overcome the limitations of classical 2D platforms. Although several different strategies have been developed to improve the angiogenesis within hydrogels, one of the main challenges in tissue engineering remains the lack of vascularization in the fabricated 3D models. The present work focuses on the high-definition (HD) bioprinting of microvascular structures directly on-chip using two-photon polymerization (2PP). 2PP is a nonlinear process, where the near-infrared laser irradiation will only lead to the polymerization of a very small volume pixel (voxel), allowing the fabrication of channels in the microvascular range (10–30 µm in diameter). Additionally, 2PP not only enables the fabrication of sub-micrometer resolution scaffolds but also allows the direct embedding of cells within the produced structure. The accuracy of the 2PP printing parameters were optimized in order to achieve high-throughput and HD production of microfluidic vessel-on-chip platforms. The spherical aberrations stemming from the refractive index mismatch and the focusing depth inside the sample were simulated and the effect of the voxel compensation as well as different printing modes were demonstrated. Different layer spacings and their dependency on the applied laser power were compared both in terms of accuracy and required printing time resulting in a 10-fold decrease in structuring time while yielding well-defined channels of small diameters. Finally, the capacity of 2PP to create vascular structures within a microfluidic chip was tested with two different settings, by direct embedding of a co-culture of endothelial- and supporting cells during the printing process and by creating a supporting, cell-containing vascular scaffold barrier where the endothelial cell spheroids can be seeded afterwards. The functionality of the formed vessels was demonstrated with immunostaining of vascular endothelial cadherin (VE-Cadherin) endothelial adhesion molecules in both static and perfused culture.

Highlights

Three-dimensional (3D) cell culture models that have the capacity to recapitulate the biochemical functionalities, mechanical properties and the microarchitecture of organs have been gaining increasing attention in biomedical research
The capacity of 2. Two-photon polymerization (2PP) to create vascular structures within a microfluidic chip was tested with two different settings, by direct embedding of a co-culture of endothelial- and supporting cells during the printing process and by creating a supporting, cell-containing vascular scaffold barrier where the endothelial cell spheroids can be seeded afterwards
UV embedding of RFP-human umbilical vein endothelial cells (HUVEC) with supporting cells in thiol-ene hydrogel Thiol-ene photo-click gelatin hydrogels already showed remarkably high processability, biocompatibility and supported the adhesion and proliferation of cells when used as a bioink for HD bioprinting [18]

Summary

Introduction

Three-dimensional (3D) cell culture models that have the capacity to recapitulate the biochemical functionalities, mechanical properties and the microarchitecture of organs have been gaining increasing attention in biomedical research. These systems could provide more insights into the pathological and physiological functions of tissues compared to 2D cell cultures [1, 2]. Natural hydrogels are physically or chemically crosslinked polymer networks which can take up large quantities of water without dissolving [6, 7] They are often derived from the non-cellular compartment of the tissues called the extracellular matrix (ECM) [8]. Thiol-ene based step-growth polymerization offers several advantages over the more traditional chain-growth hydrogels, such as the lack of oxygen inhibition and faster reaction kinetics enabling the reproducible production of 3D printed structures with high structural integrity at low light intensities [18]

Methods

Results

Conclusion