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

细胞培养实验在试图研究生物机制时提供了独特的优势，这主要归结为在实验装置中以体内实验不允许的方式同时操纵关键细胞参与者及其环境的能力。这种加强的控制源于这样一个事实，即细胞培养系统天生就不像体内培养系统那样复杂。这种复杂性的降低既是这些平台的优势，也是一个关键的限制，近年来，我们目睹了大量新技术和工具的引入，以增加细胞培养实验的复杂性。其中包括：干细胞分化和重新编程以获得正确的关键角色，实时成像技术来监控它们的动态行为和相互作用，甚至生物工程技术来控制它们在2D和3D的环境。多亏了这些，我们现在可以开始进行更复杂的实验，更好地概括我们想要研究的生物过程，但我们仍然受到技术能力的限制：i)可靠地构建具有确定结构的复杂细胞排列，ii)直接控制和观察不同类型的细胞和细胞外基质(ECM)之间的相互作用，跨越空间(即它们的相对位置)和时间(即它们在实验期间如何跨不同时间点的行为变化)iii)有效地操纵这些复杂的培养物，这些复杂的培养物本身与复杂性成反比，并且有效地分离复杂培养物的相关亚群进行分析，而不会丢失有关它们位置和相互作用的信息。为了克服这些限制并研究动态生物复杂系统，如神经电路、发育微环境、肿瘤微环境或细菌生物膜，我们需要新的平台，允许我们首先构建活细胞和细胞外基质的复杂结构，然后以动态的方式影响它们的行为和组成，最后将它们分离到相应的亚群进行分析。由于生物系统本质上是动态的，我们需要实现所有这些，同时监测培养的行为和变化。这些能力目前不存在于单一的综合平台中，但必要的技术以i)实时成像平台、ii)细胞定位工具、iii)生物绘图仪或3D生物打印机和iv)基于微流控的细胞分选器的形式提供。特别是，目前的技术差距表现为这样一个事实，即尽管商业3D生物打印机和生物绘图仪可以构建复杂的培养物，但它们不允许获取实时显微镜数据，并且不能作为动态操作工具，因为它们构建的培养物没有直接反馈。虽然可以观察其动态行为的实时成像系统可用，但没有一个单一的平台允许直接构建或操作生物打印的活细胞结构，同时获取细胞行为的实时数据。该项目的目的是利用这些不同的技术，并将它们集成到一个单一的原型平台中，我们将其命名为成像驱动多功能生物绘图仪(IDMB)。该系统将是一个开源平台，适用于任何生物实验室，并将整合实时成像系统和细胞生物绘图仪/操纵系统的功能，以提供绘制复杂排列的活细胞和细胞外基质的可能性，以及在成像数据的直接指导下以动态方式操纵和分析这些复杂的培养物。在这个项目中，我们将构建和优化IDMB系统的全功能原型，并获得其应用于体外建模的关键原则证明数据。

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.