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

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