Developing a High-throughput Single-cell Analysis Platform for Intra- and Inter-cellular Signalling in Colonic Air Liquid Interface Organoids

准确的仿生模型对于我们理解健康和疾病组织的功能是至关重要的。气液界面类器官（ALIs）最近成为一种强大的仿生组织模型[1,2]。不像传统的类器官只包含一种细胞类型(如：（上皮细胞），ALI类器官包括来自组织的所有细胞类型，包括自体免疫细胞。因此，ALI类器官可用于研究组织中所有细胞类型的交流方式。不幸的是，现有的ALI类器官培养格式笨重，吞吐量极低，并且与许多单细胞分析技术不兼容。我们假设ALI培养物可以重新设计成小型化格式，以适应高通量多路单细胞分析技术。在这个LIDo博士项目中，我们将重新设计结肠ALI类器官，从庞大的6孔格式转变为高通量的96孔板格式，然后使用我们最近开发的定制多路巯基类器官原位条形码（TOBis）质量细胞计数（MC）技术平台[4]来研究结肠组织中存在的细胞类型特异性细胞内通信信号网络。我们将进一步利用基于分裂池连接的转录组测序（SPLiT-seq）、b[5]单细胞rna测序（scRNA-Seq）配体和受体表达配对来补充这些研究，以揭示ALI类器官中所有细胞之间的细胞间信号通路。通过整合MC、scRNA-Seq和ALI类器官，该项目将为研究组织内和细胞间信号传递提供一个新的、最先进的技术平台。该项目将涵盖4个目标：目标1：从人类结肠组织中建立新的小型化生物工程ALI培养物，用于高通量应用。目的2：通过tobismc和SPLiT-Seq对ALI培养中上皮细胞、基质细胞和自身免疫细胞的同步单细胞信号量化。目标3：利用单细胞数据建立集落组织生物学的细胞内和细胞间综合计算信号模型。目的4：通过实验干扰ALI类器官中的关键信号节点，以确定组织中功能性的细胞间信号传导。

Accurate biomimetic models are essential for our understanding of how healthy and diseasedtissues function. Air liquid interface organoids (ALIs) have recently emerged as powerfulbiomimetic tissue models [1, 2]. Unlike traditional organoids that only contain one cell-type(e.g., epithelial cells), ALI organoids comprise all cell-types from a tissue - includingautologous immune cells. As a result, ALI organoids can be used to study how all cell-types ina tissue communicate. Unfortunately, existing ALI organoid cultures formats arecumbersome, extremely low throughput, and incompatible with many single-cell analysistechnologies [3].We hypothesise that ALI cultures can be reengineered into a miniaturised format to becompatible with high-throughput multiplexed single-cell analysis technologies.In this LIDo PhD project, we will reengineer colonic ALI organoids from a bulky 6-well formatinto a high-throughput 96-well plate format and then use our recently developed custommultiplexed thiol organoid barcoding in situ (TOBis) mass cytometry (MC) technologyplatform [4] to study cell-type-specific intracellular communication signalling networkspresent in colonic tissue. We will further compliment these studies with split-pool ligationbasedtranscriptome sequencing (SPLiT-seq) [5] single-cell RNA-sequencing (scRNA-Seq)ligand and receptor expression pairing to reveal intercellular signalling pathways between allcells in ALI organoids. By integrating MC, scRNA-Seq, and ALI organoids, this project willgenerate a new state-of-the-art technology platform for studying intra- and inter-cellularsignalling in tissues.The project will cover 4 Aims:Aim 1: Establish new miniaturised bioengineered ALI cultures from human colonic tissue forhigh-throughput applications.Aim 2: Concurrent single-cell signalling quantification of epithelial, stromal, and autologousimmune cells in ALI cultures by TOBis MC and SPLiT-Seq.Aim 3: Develop integrated intra- and inter-cellular computational signalling model of colonictissue biology from single-cell data.Aim 4: Experimentally perturb key signalling nodes in ALI organoids to define functional intercellularsignalling in tissues.