Periodic Reporting for period 4 - Bio-Plan (Bio-Inspired Microfluidics Platform for Biomechanical Analysis)
Reporting period: 2024-04-01 to 2024-09-30
Biomechanical interactions between cells and their environment are essential in almost any biological process, from embryonic development to organ function to diseases. Hence, biomechanical interactions are crucial for health and disease. Examples are hydrodynamic interactions through fluid flow, and forces acting directly on cells. Existing methods to analyze and understand these interactions are limited however, since they do not offer the required combination of precisely controlled flow and accurate applying and sensing of forces. Also, they often lack a physiological environment. Thus, we need a breakthrough in biomechanical analysis platform to study biomechanical interactions, to better understand the development of organ tissues as well as diseases like cancer or fibrosis, which eventually can result in novel, effective therapies and treatments.
The overall objective of the project Bio-Plan was to realize a novel microfluidic platform for biomechanical analysis with unprecedented possibilities of controlling fluid flow and applying time-dependent forces at subcellular scales in controlled environments, while also allowing for live microscopy of cellular events. We aimed to address three fundamental questions: 1. How do hydrodynamic interactions steer cellular and particle transport? 2. How do local and dynamic mechanical forces on cells fundamentally influence their behavior? 3. How do hydrodynamic interactions between cilia steer embryonic development?
The overall objective of the project Bio-Plan was to realize a novel microfluidic platform for biomechanical analysis with unprecedented possibilities of controlling fluid flow and applying time-dependent forces at subcellular scales in controlled environments, while also allowing for live microscopy of cellular events. We aimed to address three fundamental questions: 1. How do hydrodynamic interactions steer cellular and particle transport? 2. How do local and dynamic mechanical forces on cells fundamentally influence their behavior? 3. How do hydrodynamic interactions between cilia steer embryonic development?
We realised and experimentally demonstrated a biomechanical platform analysis based on magnetic artificial cilia (MAC). These were inspired by biological cilia, microscopic hairs ubiquitously present on cell surfaces, acting both as actuators and sensors, essential for swimming of microorganisms, transport of dirt out of our airways, and sensing of sound. We developed two complementary magnetically responsive artificial cilium types: microscopic magnetic artificial cilia (microMAC, Fig. WP1a) with a length of hundreds of micrometers and a radius of typically 25 micrometers, and nanoscopic magnetic artificial cilia (nanoMAC, see WP1b) with a length as small as 6 micrometers and a radius of 200 nanometers. These latter artificial cilia are as small as the smallest biological cilia found in nature. To realize motion by the magnetic cilia, we designed and realized several magnetic actuation setups. Finally, we achieved to make an integrated platform, fitting into an incubator, and we carried out biological experiments while observing live the response of the cells grown on carpets of nanoMAC.
We demonstrated that our MAC could generate substantial fluid flow in a controlled fashion (Fig. WP1c), and that we can use them as microfluidic flow sensors. Also, we showed that the MAC can be used to clean surfaces (Fig. WP2a), to transport particles in a controlled manner (Fig. WP2b), and even to create anti-biofouling surfaces (Fig. WP2c). In this work, we have combined experimental studies with numerical simulations to obtain thorough understanding of the mechanisms at work. This answered question 1.
We have cultured cells on the carpets of nanoMAC. We proved that the nanoMAC are biocompatible, and that the cell behaviour depends on the properties of the cilia (size, spacing), indicating we can indeed steer cell behaviour with our cilia (Fig. WP3a). A next important step was to carry out cell experiments while actuating the nanoMAC, for dynamic mechanotransduction analysis (WP3b). We have shown that we can do live cell microscopy and measure relevant biomarkers during actuation, and the results show that the cells indeed respond specifically to time-dependent mechanical cues. These results prove the unique capabilities of our platform – this opens a whole world of new analysis possibilities for the future. This answered question 2.
Finally, based on the unique properties of our nanoMAC we have been able to create artificial embryonic nodes using combinations of active and passive nanoscopic cilia, and we have carried quantitative flow characterisation of the embryonic flow generated by these cilia (Fig. WP4a); in parallel, we have carried out advanced numerical simulations of the cilia-driven flow in the artificial embryonic node (WP4b). The combined results of these experiments and simulations have resulted in novel insights into the effect of ciliary flow on embryonic development, contributing to a long-standing debate (WP4c). Our main finding is that the deformation of mechanosensing passive cilia, caused by flow generated by active cilia, is the most important contribution to the effect and drives the development of left-right body symmetry during embryonic development. This answered question 3.
Next to these results, the research of Bio-Plan has generated results we had not anticipated or planned in advance, in different areas, including algae growth methods (Fig. +a), a lung clearance model (Fig. +b), microrobots (Fig. +c), and micromixing. However, the most important one was the of a novel method to create very small tubular lumens in microfluidic devices, enabling to mimic small blood vessels or other tubular structures such as renal tubules (Fig. +d). This technology will enable us to create organ-on-chip systems with relevant applications in the future, such as organ models for cancer research.
Dissemination: the project has up to now resulted in 16 peer-reviewed publications in scientific journals, and at least 5 more papers are in review or in preparation. Also, results of our work have been presented as 26 oral or poster presentations at international scientific conferences, including 7 invited lectures.
Exploitation: as a follow-up, we received funding for an ERC Proof-of-Concept project, for development towards commercial implementation of micropumps based on magnetic artificial cilia. In parallel, we have founded a spin-off company on the technology, ARTIC, that will exploit this further.
We demonstrated that our MAC could generate substantial fluid flow in a controlled fashion (Fig. WP1c), and that we can use them as microfluidic flow sensors. Also, we showed that the MAC can be used to clean surfaces (Fig. WP2a), to transport particles in a controlled manner (Fig. WP2b), and even to create anti-biofouling surfaces (Fig. WP2c). In this work, we have combined experimental studies with numerical simulations to obtain thorough understanding of the mechanisms at work. This answered question 1.
We have cultured cells on the carpets of nanoMAC. We proved that the nanoMAC are biocompatible, and that the cell behaviour depends on the properties of the cilia (size, spacing), indicating we can indeed steer cell behaviour with our cilia (Fig. WP3a). A next important step was to carry out cell experiments while actuating the nanoMAC, for dynamic mechanotransduction analysis (WP3b). We have shown that we can do live cell microscopy and measure relevant biomarkers during actuation, and the results show that the cells indeed respond specifically to time-dependent mechanical cues. These results prove the unique capabilities of our platform – this opens a whole world of new analysis possibilities for the future. This answered question 2.
Finally, based on the unique properties of our nanoMAC we have been able to create artificial embryonic nodes using combinations of active and passive nanoscopic cilia, and we have carried quantitative flow characterisation of the embryonic flow generated by these cilia (Fig. WP4a); in parallel, we have carried out advanced numerical simulations of the cilia-driven flow in the artificial embryonic node (WP4b). The combined results of these experiments and simulations have resulted in novel insights into the effect of ciliary flow on embryonic development, contributing to a long-standing debate (WP4c). Our main finding is that the deformation of mechanosensing passive cilia, caused by flow generated by active cilia, is the most important contribution to the effect and drives the development of left-right body symmetry during embryonic development. This answered question 3.
Next to these results, the research of Bio-Plan has generated results we had not anticipated or planned in advance, in different areas, including algae growth methods (Fig. +a), a lung clearance model (Fig. +b), microrobots (Fig. +c), and micromixing. However, the most important one was the of a novel method to create very small tubular lumens in microfluidic devices, enabling to mimic small blood vessels or other tubular structures such as renal tubules (Fig. +d). This technology will enable us to create organ-on-chip systems with relevant applications in the future, such as organ models for cancer research.
Dissemination: the project has up to now resulted in 16 peer-reviewed publications in scientific journals, and at least 5 more papers are in review or in preparation. Also, results of our work have been presented as 26 oral or poster presentations at international scientific conferences, including 7 invited lectures.
Exploitation: as a follow-up, we received funding for an ERC Proof-of-Concept project, for development towards commercial implementation of micropumps based on magnetic artificial cilia. In parallel, we have founded a spin-off company on the technology, ARTIC, that will exploit this further.
Many of the achievements of Bio-Plan advance the field beyond the state of the art, such as the high level of fluid flow and particle manipulation control by artificial cilia, the combination of artificial cilia with biological cell culture, and the anti-biofouling mechanism by artificial cilia, and the novel insights in embryonic development and response of cells to time-dependent mechanical cues. However, the largest step beyond state of the art has been the development of the nanoscopic highly motile artificial cilia, with sizes as small as the smallest biological cilia. Additionally, the novel method to create very small tubular lumens in microfluidic devices, not foreseen initially, forms a breakthrough for organ-on-chip models. All these achievements together form the basis of future research, in which we will apply the platform we developed to understand the effects of mechanical cues on the development of diseases like cancer, which eventually can result in novel, effective therapies and treatments.