Particle distribution dynamics in nonlinear bifurcating networks

Project Information

BiNet

Grant agreement ID: 101043314

DOI

10.3030/101043314

EC signature date 30 May 2022

Start date 1 October 2022

End date 30 September 2027

Funded under

• European Research Council (ERC)

Total cost

€ 2 000 000,00

EU contribution

€ 2 000 000,00

Coordinated by

OULUN YLIOPISTO
Finland

Periodic Reporting for period 1 - BiNet (Particle distribution dynamics in nonlinear bifurcating networks)

Reporting period: 2022-10-01 to 2025-03-31

Summary of the context and overall objectives of the project

Bifurcating networks are ubiquitous in nature such as vasculature/pulmonary networks, kidney urinary tract, and branching in plants. In addition to their role of transporting the carrying fluid, these networks distribute discrete particles suspended in the fluid leading to intricate spatiotemporal particle flow dynamics. The heterogenous distribution of red blood cells (RBC) in microcirculation is an example of this behaviour, consequences of which are not well-understood. Our objective is to explain the fundamental principles and implications of nonuniform particle distributions in bifurcating networks using RBC flow in vasculature as a model system. Currently there is no systematic approach to study such complex particle distribution dynamics. We use droplet microfluidic as an analogue of the biological network. Microfluidics provide superb control of the droplets/particles, carrying fluid and
network properties in highly engineered microfabricated devices. We aim to understand RBC distribution patterns in capillary network and the consequences during vascularization and organogenesis. Our approach is (i) to observe the in vivo RBC fractionation in chick embryo vasculature, (ii) to develop its in vitro analogue using droplet microfluidics, (iii) to develop in silico model and determine the governing parameters. This project will discover the foundations of particle transport phenomena in nonlinear bifurcating networks and address the long-lasting question of RBC nonuniformity in microcirculation and its implications as a groundbreaking contribution. Another key outcome is the correlation between RBC heterogeneity to corresponding organ growth by visualising RBC distrubtions at single cell resolution and monitoring vasculogenesis of the network simultaneously. With this approach, we aim to develop a fundamental understanding of vascularization dynamics as well as the implications of non uniform RBC distribution in tissue development. Additionally, the project develops several tools and techniques for vascularized organoid development and organ-on-chip systems.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Our work so far has focused on developing the analytica, numerical tools and developing the techniques for in vivo vascularization monitoring. We have developed a microfluidic technique for in vitro vascularization using xenotransplantation. To the best of our knowledge, this is the first demonstration of an in vivo vascularized transplanted tissue being detached from the living organism and later perfused in vitro. The dynamics of vascularization is still not well-understand and it is the core question we are tackling in our project. While observing embryonic development for extended times, we were also able to develop this microfluidic organ-on-chip system for kidney organoid vascularization. This system provides a solution for in vitro vascularized organoids, specifically kidney towards artificial kidney development.
The second achievement in the project is the oscillating hypoxia model that can be embedded to commercial organoid culturing platforms mimic real-world hypoxia conditions. We verified the sensitivity of cells adjacent to the microchannel to fluctuations of oxygen content in the microchannel as we monitored and calculated the frequency of oxygen probe signal oscillations. The calculated frequency and period of oscillations in the oxygen probe signal, correspond to the frequency and period of oxygen oscillations in the microchannel. Moreover, by monitoring the reactive oxygen species (ROS) production after each cycle of hypoxia/reoxygenation we further validated the reliability of our model to study the cycling hypoxia implication. To our knowledge, this is the first in vitro model, that can be an alternative to existing in vitro and in vivo models, that provides experimental evidence into the effect of heterogenous oxygen supply on pericapillary tissue, the swiftness of cellular response to oxygen fluctuations, regional blood flow regulation and red blood cell distribution.
The third achievement in the project is the numerical model we developed to simulate spontaneously generated RBC concentration fluctuations. Using this tool and the in vitro droplet model, we were able to demonstrate that RBC distritubtion in networks can be generating spontanenous oscillations which can explain the over-vascularization followed by pruning during embryonic development. This simulation generates two outputs: Droplets, a variable that contains comprehensive information about each droplet's position, and Evolution in Number of Droplets, which records how droplet distributions vary over time across channels. These results are critical for understanding flow dynamics, investigating oscillatory phenomena, and the implications of non-uniform RBC distributions.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

So far, our main result beyond the state-of-the-art is the in vitro microfluidic vascularization system we developed. There is significant need for in vitro vascularized organoids with the long-term aim of obtaining artificial tissues. Although our cell co-culturing abilities have developed significantly, vascularization of these constructs is still the major problem. To address this problem we develoepd a microfluidic system that can be transplanted on a chiekcen embryo chorioallantoic membrane to initiate vascular development. After maturation of the vessels, we were able to remove this construct from the chicken to the bench and continue perfusion. During this process we were able to monitor the RBC flow and development of the tissue. The blood circulation was restored in the organoid by external perfusoin and vasculature development continued. To our knowledge, this is the first demonstration of the combination of in vivo and in vitro approaches for vascularized organoid development. Existing in vitro microfluidic approaches only provide initial vascularization of organoids and fail to generate in vivo like networks. On the other side,in vivo transplantation to animal models is problematic due to throughput and ethical concerns. Our solution provides an alternative to both of these approaches by xenotransplantation onto a chicken embryo only for vasuclar initiation phase, which can than later be perfused and grown in vitro under laboratory conditions.

The three main developments in the project, in silico, in vitro and in vivo models