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，从农业到粮食生产和人类健康。微生物群描述了共生微生物共存并与宿主生物相互作用的微环境，是一个复杂而动态的生态系统，难以在实验室中重建。这主要是由于微生物比哺乳动物细胞生长得快得多，这将很快压倒培养物，并对哺乳动物细胞的存活产生负面影响。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-微生物研究中这一未被满足的技术差距，这些培养系统可以控制微生物在受限液滴阵列中的生长，这些液滴阵列在物理上是分离的，但仍与周围的哺乳动物细胞保持化学连接。在这个项目中，我们的目标是在一个直接接触人类上皮细胞的细菌菌落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）的水溶液形成两个独立的不混相。这些相支持在上皮细胞层上建立细菌菌落和生物膜，细菌培养物被困在富含dex相溶液的液滴中，以防止过度生长到周围富含peg的培养基中。然而，这种原型配方并不理想，也不为人所知，因此对哺乳动物细胞活力产生了显著的负面影响，对微生物的毒性尚不清楚。在这里，我们的目标是设计一个家族的生物相容性AMPS形成。我们将筛选广泛的聚合物以选择那些具有生物相容性的聚合物。理想的AMPS组合物还应该最大限度地减少对广谱分泌生物分子的扩散障碍，包括信号肽、激素和代谢物。候选AMPS制剂中生物分子的分配系数将使用先进的蛋白质组学分析进行评估。最后，候选制剂将使用已知的信号通路进行调整，以确保在细菌菌落和哺乳动物细胞之间建立间接的、原生的细胞-细胞通信。该项目将为我们对生物相容性相分离系统的理解奠定基础，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.