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。气液界面有机化合物(ALIS)是近年来出现的一种强有力的仿生组织模型[1，2]。与只包含一种细胞类型(如上皮细胞)的传统器官不同，ALI器官包括组织中的所有细胞类型--包括自体免疫细胞。因此，ALI类有机化合物可以用来研究组织中所有类型的细胞是如何沟通的。不幸的是，现有的ALI有机物培养格式繁琐，吞吐量极低，并且与许多单细胞分析技术不兼容[3]。我们假设ALI培养物可以被重新工程成一种小型化的格式，以适应高通量的多路单细胞分析技术。在这个LIDO PHD项目中，我们将把结肠ALI有机物从笨重的6孔格式重新设计成高通量的96孔板格式，然后使用我们最近开发的定制多路硫醇有机物原位条码(TOBIS)质量细胞术(MC)技术平台[4]来研究结肠组织中特定细胞类型的细胞内通讯网络。我们将进一步通过基于裂池连接的转录组测序(Split-Seq)[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ALL细胞之间的细胞间信号通路。通过整合MC、scRNA-Seq和ALI有机体，该项目将产生一个新的最先进的技术平台，用于研究组织中的细胞内和细胞间信号传递。该项目将包括4个目标：目标1：建立新的用于高通量应用的微型人结肠组织生物工程ALI细胞。目标2：利用Tobis MC和Split-Seq对ALI培养中的上皮、基质和自体免疫细胞进行并发单细胞信号定量。目标3：利用单细胞数据建立集成的结肠组织生物学细胞内和细胞间信号传递模型。目标4：实验扰动ALI细胞器中的关键信号节点，以确定组织中功能的细胞间信号传递。

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.