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

微生物遍布自然界，涉及人类生活的方方面面，从农业到食品生产和人类健康。微生物组描述了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，难以在实验室中重现。这主要是由于微生物的生长速度比哺乳动物细胞快得多，这将迅速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个基于聚合物的培养系统家族来解决宿主微生物研究中这一未满足的技术差距，该系统可以控制物理分离的有限液滴阵列中微生物的生长，但仍然与周围的哺乳动物细胞化学连接。在这个项目中，我们的目标是在由与人类上皮细胞直接接触的细菌菌落组成的模型中开发水性多相系统（AMPS）制剂。在以前的研究中，我们已经证明，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溶相。这些相支持在上皮细胞层上建立细菌菌落和生物膜，其中细菌培养物被捕获在富含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，这种原型制剂并不理想，也没有得到很好的理解，因此对哺乳动物细胞活力造成了显著的负面影响，其对微生物的毒性也是未知的。在这里，我们的目标是设计一个家庭的生物相容性AMPS形成。我们将筛选范围广泛的聚合物，以选择那些生物相容性。理想的AMPS组合物还应使对广谱分泌的生物分子（包括信号肽、激素和代谢物）的扩散屏障最小化。将使用先进的蛋白质组学分析评估候选AMPS制剂中生物分子的分配系数。最后，将使用已知的信号传导途径来调整候选制剂，以确保在细菌菌落和哺乳动物细胞之间建立间接的、天然样的细胞间通信。该项目将为我们理解生物相容性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.