Developing an Open Source Imaging-Driven Multifunctional Bioplotter (IDMB) for next generation in vitro modelling

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Cell culture experiments offer unique advantages when trying to study biological mechanisms, which mainly come down to the ability to manipulate both the key cell players and their environment within an experimental set-up, in ways that in vivo experiments would not otherwise allow. This increased control arises from the fact that cell culture systems are inherently less complex than their in vivo counterparts. This reduction of complexity is both an advantage and a crucial limitation of these platforms, and in recent years we have witnessed a plethora of new technologies and tools introduced to increase complexity of cell culture experiments.Amongst these: stem cell differentiation and reprogramming to obtain the right "key players", live imaging techniques to monitor their dynamic behaviour and interactions, and even bioengineering techniques to control their environment in both 2D and 3D. Thanks to these we can now start to perform more complex experiments that better recapitulate the biological processes we want to study, but we are still limited by our technical capacity to:i) reliably construct complex arrangements of cell with determined architecture, ii) directly control and observe the interactions between different cell types and extracellular matrix (ECM), both across space (i.e. their relative position) and time (i.e. how their behavior changes across different time-points during the experiment)iii) effectively manipulate these complex cultures, which inversely scales with complexity itselfiv) efficiently separate relevant subpopulation of the complex cultures for analysis without losing information on their position and interactions. To overcome these limitations and study dynamic biological complex systems like neural circuits, developmental niches, tumour microenvironments, or bacterial biofilms we need new platforms that allow us to first construct complex architectures of live cells and ECM, then influence their behaviour & composition in a dynamic way, and finally separate them into relevant subpopulation for analysis. And, as biological systems are dynamic in nature, we need to achieve all of this, while monitoring the behaviour and changes in the composition of the culture.These capabilities currently do not exist within a single integrated platform, but the technologies necessary are available in the form of i) live imaging platforms, ii) cell-positioning tools, iii) bioplotters or 3D bioprinter and iv) microfluidic-based cell sorters. Particularly, the current technological gap is represented by the fact that while commercial 3D bioprinters and bioplotters can construct complex cultures, they do not allow to acquire live microscopy data, and cannot function as dynamic manipulation tools as they construct cultures without direct feedback. And while live imaging systems that can observe their dynamic behaviour are available, no single platform allows to directly construct or manipulate bioprinted live cell constructs while at the same time acquiring live data on the cell behaviour.The aim of this project is to capitalise on these different technologies and integrate them within one single prototype platform, that we have named Imaging-Driven Multifunctional Bioplotter (IDMB). This system will be an open-source platform, adaptable to any biology lab and will integrate the functionality of live imaging systems and cell bioplotters/manipulation systems to offer the possibility of plotting live cells and ECM in complex arrangements, as well as manipulate and analyse these complex cultures, in a dynamic way, guided directly by the imaging data. During this project we will construct and optimise a fully functional prototype of the IDMB system and obtain key proof-of-principle data of its application to in vitro modelling.