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

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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.