New approaches for investigating intestinal barrier function in-vitro

The intestine has important and unique functions that are absolutely essential for maintaining whole organism health. Around 40% of the body’s energy expenditure can be attributed to regulation of the intestinal barrier between luminal contents and the body. This is exquisitely controlled to prevent malnutrition or systemic infection by commensal or pathogenic bacteria. Daily absorption of fluids (dietary and secretions) from the intestinal lumen into the body is in the region of 8 litres. To achieve efficient absorption, the intestine has a surface area of around 400m² and an epithelial layer that is only one cell thick. This process provides a huge challenge to prevent invasion by the luminal microbiome. Intestinal permeability and epithelial cell health status are particularly important regulators of intestinal barrier function and if compromised can lead to a multitude of diseases (intestinal and systemic) and inflammation. The physiological mechanisms that maintain this tightly regulated barrier remain incompletely understood and are difficult to explore owing to the lack of suitable in-vitro models.

Intestinal permeability is difficult to investigate due to a lack of suitable in-vitro assays. Cell line monolayers derived from human colon cancers on transwell filters are often used to assess transepithelial electrical resistance and flux of substances across the epithelium, but these culture systems do not recapitulate normal gut growth dynamics. Other studies consist of oral dosing of whole animals or tissue explants that are maintained in short-term culture. 

Ground-breaking research by Hans Clevers’ group has determined culture conditions for maintaining intestinal stem cell growth permitting the generation of long-lived three-dimensional organoid “mini-gut” cultures. Organoids maintain all differentiated cell types and growth dynamics observed in the intestinal epithelium in-vivo and grow with a central lumen. The apical membrane on the inside and basolateral membrane on the outer surface makes the apical pole difficult to access. Application of dietary components and bacterial products/bacteria to the apical pole of epithelial cells is important as apical and basolateral membranes act very differently to the same stimuli. Several groups including our own have developed microinjection techniques to access the lumen and apply dietary/microbiome/mycobiome components and permeability markers to assess intestinal permeability, however, these assays are time consuming and do not lend themselves to high-throughput studies.  

This year, techniques have been developed to reverse the polarity of intestinal organoid culture resulting in the apical membrane on the outside and basolateral membrane on the inside. We now want to develop high-throughput assays utilising apical-out and basal-out organoid systems to investigate how diet/genetics/age and microbiome interact to modulate intestinal permeability and barrier function.

Informal enquiries may be made to carried@liverpool.ac.uk

HOW TO APPLY

 Applications should be made by emailing bbsrcdtp@liverpool.ac.uk with a CV and a covering letter, including whatever additional information you feel is pertinent to your application; you may wish to indicate, for example, why you are particularly interested in the selected project/s and at the selected University. Applications not meeting these criteria will be rejected. We will also require electronic copies of your degree certificates and transcripts.

 In addition to the CV and covering letter, please email a completed copy of the NLD BBSRC DTP Studentship Application Details Form (Word document) to bbsrcdtp@liverpool.ac.uk, noting the additional details that are required for your application which are listed in this form. A blank copy of this form can be found at: https://www.nld-dtp.org.uk/how-apply.