Epithelial barrier model: in silico modelling and high throughput assessment
Lead Research Organisation:
University of Birmingham
Department Name: Institute of Clinical Sciences
Abstract
The project aims to enhance the functionality of our established technology to produce complex stratified epithelial organotypic rafts by increasing throughput, reproducibility and confidence to replace animal models in oral mucosa and skin research.
Human skin and oral mucosa are protected from the external environment by multi-layered complex epithelia. These epithelia form a barrier against abrasion, toxins, infectious agents, water loss and UV radiation, and as such are key for maintaining functional healthy tissues. When epithelial barrier is compromised or decreased (i.e. in ageing, during chemotherapy, or in inflamed tissues) this can lead to infections, cancer or autoimmune conditions.
Epithelial cells (also known as keratinocytes) multiply within a layer close to underlying connective tissue and then migrate towards the tissue surface. During this migration keratinocytes differentiate, increase cell-cell interactions and production of lipids, keratins and other proteins reinforcing the barrier structure in the process called maturation. The result is multi-layered, strong and constantly replenished tissue with tightly controlled permeability.
Understanding how the epithelial barrier is formed and maintained in healthy tissues is very important for addressing disease states and treatments. This knowledge cannot be advanced by using standard cell culture methods in which cells are grown in a single layer or as spheroids. Key aspects of epithelial biology including differentiation, signalling, barrier function, gene and protein expression, infections and wound healing can only be fully addressed in either animal models or 3D organotypic cultures that replicate stratified epithelial tissue in vitro. Typically, in this 3D models (epithelial rafts) keratinocytes are encouraged to form multi-layered differentiating tissue by maintaining them at air-liquid interface (ALI) on top of fibroblast-containing collagen (connective tissue equivalent).
Two aspects of epithelial organotypic rafts cultures currently prevent their wider application as well as ability to limit the use of animal models. Firstly, understanding of stratification and development of epithelial barrier in these models is far from complete. Here we will take advantage of a novel technology (single cell RNA-sequencing) to model epithelial stratification layer by layer and track barrier-related gene expression during maturation in tissue equivalents of oral epithelium. This novel approach will form a reference for future model developments.
Secondly, the lack of reliable high throughput formats prevents their use in applications requiring large numbers of replicates and controls. The high-throughput formats are difficult to execute as ALI maintenance is labour-consuming and operator-dependent, with any variation in ALI leading to inconsistent growth and stratification. To address these issues, we recently developed and tested a Buoyant Epithelial Culture Device (BECD) in which ALI is maintained automatically by the raft floating on top of media, considerably simplifying epithelial tissue generation thus reducing costs and improving reproducibility and wider adoption. In this project, we intend to evolve the BECDs towards a semi-high throughput system and adapt it to an array of methodologies to assess epithelial integrity and barrier function. Finally, to minimize the use of animal-derived reagents, we will attempt to replace collagen with synthetic hybrid hydrogels modified to replicate physical and mechanical properties of collagen gels.
Through advancing our understanding of in vitro produced epithelial tissues and enabling higher throughput for their assessment, the project aims to increase confidence in using these models in basic and translational research, putting it in favour of animal models.
Human skin and oral mucosa are protected from the external environment by multi-layered complex epithelia. These epithelia form a barrier against abrasion, toxins, infectious agents, water loss and UV radiation, and as such are key for maintaining functional healthy tissues. When epithelial barrier is compromised or decreased (i.e. in ageing, during chemotherapy, or in inflamed tissues) this can lead to infections, cancer or autoimmune conditions.
Epithelial cells (also known as keratinocytes) multiply within a layer close to underlying connective tissue and then migrate towards the tissue surface. During this migration keratinocytes differentiate, increase cell-cell interactions and production of lipids, keratins and other proteins reinforcing the barrier structure in the process called maturation. The result is multi-layered, strong and constantly replenished tissue with tightly controlled permeability.
Understanding how the epithelial barrier is formed and maintained in healthy tissues is very important for addressing disease states and treatments. This knowledge cannot be advanced by using standard cell culture methods in which cells are grown in a single layer or as spheroids. Key aspects of epithelial biology including differentiation, signalling, barrier function, gene and protein expression, infections and wound healing can only be fully addressed in either animal models or 3D organotypic cultures that replicate stratified epithelial tissue in vitro. Typically, in this 3D models (epithelial rafts) keratinocytes are encouraged to form multi-layered differentiating tissue by maintaining them at air-liquid interface (ALI) on top of fibroblast-containing collagen (connective tissue equivalent).
Two aspects of epithelial organotypic rafts cultures currently prevent their wider application as well as ability to limit the use of animal models. Firstly, understanding of stratification and development of epithelial barrier in these models is far from complete. Here we will take advantage of a novel technology (single cell RNA-sequencing) to model epithelial stratification layer by layer and track barrier-related gene expression during maturation in tissue equivalents of oral epithelium. This novel approach will form a reference for future model developments.
Secondly, the lack of reliable high throughput formats prevents their use in applications requiring large numbers of replicates and controls. The high-throughput formats are difficult to execute as ALI maintenance is labour-consuming and operator-dependent, with any variation in ALI leading to inconsistent growth and stratification. To address these issues, we recently developed and tested a Buoyant Epithelial Culture Device (BECD) in which ALI is maintained automatically by the raft floating on top of media, considerably simplifying epithelial tissue generation thus reducing costs and improving reproducibility and wider adoption. In this project, we intend to evolve the BECDs towards a semi-high throughput system and adapt it to an array of methodologies to assess epithelial integrity and barrier function. Finally, to minimize the use of animal-derived reagents, we will attempt to replace collagen with synthetic hybrid hydrogels modified to replicate physical and mechanical properties of collagen gels.
Through advancing our understanding of in vitro produced epithelial tissues and enabling higher throughput for their assessment, the project aims to increase confidence in using these models in basic and translational research, putting it in favour of animal models.
Technical Summary
In vitro tissue mimics of stratified epithelia have existed for the last 30 years and are considered the best choice to study human skin and mucosa. However, their wider uptake is hindered by technical challenges, poor reproducibility, high cost, lack of reliable high throughput systems and limited understanding of such models on molecular level.
Our proposed solution will build on simple but proven technology available in our laboratory where in vitro epithelium is established using a 3D-printed Buoyant Epithelial Culture Device (BECD) and maintained automatically in air-liquid interface (ALI) by floating on top of media. In this project, the BECDs will be adapted to semi-high throughput system while incorporating assessment of epithelial integrity and barrier function (light-sheet microscopy, transepithelial electrical resistance, tracer flux assay). Here we aim for enhancing model reproducibility and repeatability, therefore permitting its use in applications requiring large numbers of replicates and controls.
To better understand epithelial stratification and barrier formation in tissue equivalents of both keratinized and non-keratinized oral epithelia we will apply computational modelling of epithelial strata based on single cell RNA-sequencing data. This novel approach aims to increase confidence in the model and will result in establishing a point of reference for future studies, including genetic and environmental challenges.
To minimise the use of animal-derived products in the final 3D culture system the collagen component will be replaced by hybrid hydrogels tuned to replicate physical and mechanical properties of collagen-based gels.
The proposed model system increases the ease of use of the 3D epithelial rafts, is cheap and adaptable, therefore enhancing the likelihood of the technology uptake by wider research community and making it an attractive alternative for replacing the existing animal models.
Our proposed solution will build on simple but proven technology available in our laboratory where in vitro epithelium is established using a 3D-printed Buoyant Epithelial Culture Device (BECD) and maintained automatically in air-liquid interface (ALI) by floating on top of media. In this project, the BECDs will be adapted to semi-high throughput system while incorporating assessment of epithelial integrity and barrier function (light-sheet microscopy, transepithelial electrical resistance, tracer flux assay). Here we aim for enhancing model reproducibility and repeatability, therefore permitting its use in applications requiring large numbers of replicates and controls.
To better understand epithelial stratification and barrier formation in tissue equivalents of both keratinized and non-keratinized oral epithelia we will apply computational modelling of epithelial strata based on single cell RNA-sequencing data. This novel approach aims to increase confidence in the model and will result in establishing a point of reference for future studies, including genetic and environmental challenges.
To minimise the use of animal-derived products in the final 3D culture system the collagen component will be replaced by hybrid hydrogels tuned to replicate physical and mechanical properties of collagen-based gels.
The proposed model system increases the ease of use of the 3D epithelial rafts, is cheap and adaptable, therefore enhancing the likelihood of the technology uptake by wider research community and making it an attractive alternative for replacing the existing animal models.
