Speaker
Description
Introduction
Despite significant developments in endothelial-cell (EC) manipulation techniques, a proper in vitro model of a functional microvasculature with controlled local interconnectivity under well-defined global architecture is still lacking. Here, we report the generation of such controlled multi-scale vascular networks via manipulation of tens of sprouting EC ‘seeds’. We exploit magnetic patterning to assemble EC-coated superparamagnetic microbeads into ordered arrays and establish effective growth rules governing the development of interconnectivity and directionality of the networks depending on the applied seed-seed spacing.
Methods
Our microtissue-assembly method relies on the use magnetic hedgehog-like templates—arrays of permanent neodymium micromagnets arranged underneath a cell culture chamber—which guide the assembly of paramagnetic EC-coated beads on the fibrin-filled chip into a pre-designed quasi-2D pattern. Under long-term culture, the EC-seeds develop radial protrusions or ‘sprouts’ extending into the surrounding extracellular-matrix(ECM)-mimicking fibrin hydrogel and interconnect to form a percolated network. We use a numerical workflow that we introduced previously [1] to further develop a dedicated custom image processing software allowing multi-parametric morphological and topological characterization of the system of multiple sprouting and interconnecting microvasculatures.
Results
We demonstrated that the spatial organization of the microvasculature can be controlled at both microscopic and mesoscopic level via directed-assembly of EC-microcarriers. We use our system to establish a critical seed-seed spacing below which the neighboring microvasculatures become interconnected, and above which they remain disconnected, even at late times of culture. In the latter case, they can be treated as practically independent which allows systematic monitoring of multiple EC-seeds developing in nearly identical conditions. Developed microvascular networks exhibit characteristics of mature, lumenized vessels. We show that the microvascular arrays co-cultured, e.g., with cancer cells (HeLa in our case), can efficiently serve as a high-throughput platform for functional high-content screening of various anti-angiogenic compounds in full 3D microenvironment. In this respect, our system allows the measurement of a range of morphological and network-topological parameters unavailable with more conventional angiogenesis or vasculogenesis assays.
We showed that the EC-seed-based approach offers a range of advantages over conventional EC-manipulation techniques including: (i) expedited sprouting, (ii) spatial control over interconnections, (iii) reduction in cell consumption by >100x, and (iv) native high-throughput format. We validated our method using both human umbilical vein endothelial cells (HUVECs) as well as human induced pluripotent stem cells (hiPSC)-derived endothelial cells.
Discussion
The magnetic field-driven pre-patterned microvascular arrays offer a uniquely precise and standardized vascular-microtissue engineering tool with broad applications, ranging from angiogenesis research to high-throughput drug testing, and with possible extension to organ-on-chip and tissue-regenerative approaches.
References
[1] Rojek, K.O., et al., Long-term day-by-day tracking of microvascular networks sprouting in fibrin gels: From detailed morphological analyses to general growth rules. APL Bioengineering, 2024, 8(1), 016106.
This work was supported by grants Sonatina (Grant No. 2020/36/C/NZ1/00238 awarded
to K.O.R.) and Opus (Grant No. 2022/45/B/ST8/03675 awarded to J.G.) from the Polish
National Science Center (NCN).
53381519688