Design of liquid scaffolds based on aqueous multi-phase systems (AMPS) for co-culture of microbes and mammalian cells

微生物在自然界中随处可见，涉及人类生活的方方面面，从农业到食品生产和人类健康。微生物群描述了共生微生物与宿主生物共存并相互作用的微环境，是一个复杂而动态的生态系统，很难在实验室中重建。这主要是因为微生物的生长速度远远快于哺乳动物细胞，这将很快压倒培养物，并对哺乳动物细胞的生存产生负面影响。拟议的研究计划将通过创建一系列基于聚合物的培养系统来解决宿主-微生物研究中尚未填补的技术差距，这些系统可以控制受限液滴阵列中微生物的生长，这些液滴阵列物理上是分开的，但仍与周围的哺乳动物细胞保持化学联系。在这个项目中，我们的目标是在一个由细菌菌落与人类上皮细胞直接接触的模型中开发水相多相系统(AMPS)配方。在以前的研究中，我们已经证明了含有聚乙二醇(PEG)和葡聚糖(DEX)的水溶液形成了两个独立的不相容相。这些阶段支持在上皮细胞层上建立细菌菌落和生物膜，在那里细菌培养被困在富含DEX的相溶液的“液滴”中，以防止过度生长到周围富含聚乙二醇的介质中。然而，这一原型配方并不理想，也不为人所熟知，因此对哺乳动物细胞活性产生了显著的负面影响，其对微生物的毒性尚不清楚。在这里，我们的目标是设计一种生物相容的安培形成。我们将筛选范围广泛的聚合物，以选择那些生物相容的聚合物。理想的AMPS组合物还应该最小化对广泛分泌的生物分子的扩散障碍，包括信号肽、激素和代谢物。生物分子在候选AMPS配方中的分配系数将使用高级蛋白质组学分析进行评估。最后，将使用已知的信号通路来调整候选配方，以确保在细菌菌落和哺乳动物细胞之间建立间接的、天然的细胞-细胞通信。该项目将为我们理解生物相容的相分离系统奠定基础，并有助于未来先进培养平台的设计。

Microbes are found throughout nature and are involved in every facet of human lives, from agriculture, to food production and human health. The microbiome, which describes the microenvironment where commensal microbes coexist and interact with host organism, is a complex and dynamic ecosystem that is difficult to recreate in the laboratory. This is mostly due to the fact that microbes grow much faster than mammalian cells, which will quickly overwhelm the culture and negatively affect mammalian cell survival. The proposed research program will address this unmet technological gap in host-microbe research by creating a family of polymer-based culture systems that can control the growth of microbes in confined droplet arrays that are physically separated, but remains chemically connected to surrounding mammalian cells. In this project, we aim to develop aqueous multi-phase system (AMPS) formulations in a model consisting of bacteria colonies in direct contact with human epithelial cells. In previous studies, we have demonstrated that aqueous solutions containing polyethylene glycol (PEG) and dextran (DEX) form two separate immiscible phases. These phases support the establishment of bacterial colonies and biofilms over an epithelial cell layer, where bacteria cultures are trapped in ‘droplets' of DEX-rich phase solution to prevent overgrowth into surrounding PEG-rich medium. However, this prototype formulation was not ideal and well understood, thus posing significant negatively effects on mammalian cell viability and its toxicity toward microbes is unknown. Here we aim to design a family of biocompatible AMPS formation. We will screen a wide range of polymers to select those that are biocompatible. The ideal AMPS composition should also minimize diffusion barrier towards a wide spectrum of secreted biomolecules, including signalling peptides, hormones, and metabolites. Partitioning coefficients of biomolecules in candidate AMPS formulations will be evaluated using advanced proteomic analysis. Finally, candidate formulation will be tuned using known signalling pathways to ensure the establishment of indirect, native-like cell-cell communications between bacteria colonies and mammalian cells. This project will build the foundation for our understanding of biocompatible phase separation systems and aid future designs of advance culture platforms.