3D in vitro systems are an envisioned alternative to animal models especially in drug testing for research of antitumor treatments (1). Within native tissue microenvironments, the vascular system supports the physiological organ growth with nutrients and growth factors, but also plays important role in pathological conditions such as treatment-resistant tumor progression. Among other cancer types, malignant pleural mesothelioma (MPM) is an example of the latter implication. Reproducing vascular organization within in vitro models of cancer is therefore highly needed for more reliable in vitro drug testing platforms. Therefore, bottom-up approaches and novel biofabrication methods have been increasingly considered for the creation of functional tissues in a layer-by-layer approach. Sound patterning allows the spatial arrangement of biological materials such as individual cells or spheroids (2). The sound-driven hydrodynamic forces induce contactless condensation of cells within hydrogels into defined and reproducible patterns (3). This fast, mild, and simple process can be applied to assemble endothelial cells into a microcapillary network which could serve as vascularization system for functional 3D models at the centimeter scale.
Green fluorescent protein expressing human umbilical vein endothelial cells (gfp-HUVEC), human pericytes (hPC) from placenta were used for sound patterning of the microcapillary network layer in fibrin. The patterning procedure was realized with the sound patterning device (mimiX biotherapeutics, Switzerland). The microcapillary network was characterized with fluorescence microscopy. A proof-of-concept of tumor microenvironment (TME) was realized by adding a heterotypic tumor spheroid to the assembled microcapillary network. MPM cells and human fibroblast were used for the tumor spheroid preparation by spontaneous assembly in low adhesion well plates. The growth of the microcapillary network was quantified from the fluorescence image analysis. Ultimately, the effects of anticancer (Cisplatin, Platinol®) and antiangiogenic (Bevacizumab, Avastin®) drugs on the model were evaluated.
Sound-patterned microcapillary ring networks were created. The high cell packing density induced by sound into the pattern's line facilitated cell-cell connection to form microcapillary structures with the expression of VE-cadherin and lumen formation. The microcapillary ring pattern had a diameter of 1781 ± 142 µm and a thickness depending on the cell seeding density (100-400 ± 30 μm). The presence of the tumor spheroids induced 100 % more area covered by the network compared to the capillary network cultured alone, and further 50 % increase in presence of anticancer drug and tumor spheroid.
We demonstrated that a microcapillary network layer can be fabricated by sound patterning technology. Thereby, we created a 3D in vitro model used to assess the crosstalk between different biological components as proven by drug-induced biological response. Overall, in this study a novel concept for biofabrication of vascularized models is presented as an alternative to overcome present limitations. In subsequent studies, the system will be transferred to a custom-built open chamber to host a centimeter scale multicellular construct, allowing for perfusion of the vascular network.
1) S. Perrin et al. Nature, 2014
2) D. Petta et al. Biofabrication, 2020
3) A.G. Guex et al. Materials Today Bio, 2021