Development of a lab-on-a-chip to potentially model a driver of colon carcinogenesis. (2020)
Type of ContentTheses / Dissertations
Thesis DisciplineBiological Sciences
Degree NameMaster of Science
PublisherUniversity of Canterbury
AuthorsThomas, Annabella Fayeshow all
This thesis describes the design and fabrication of a microfluidic lab-on-a-chip (LOC) device that mimics the lumenal architecture of the human gut and was used to investi- gate a bacterial toxin as a driver of colon carcinogenesis. A lumen was created inside a hydrogel within a PDMS LOC device using a PDMS rod to enable adherence of human colonic HT29 cells. To enable this, the hydrophobicity / hydrophilicity of three different concentration gelatin and collagen hydrogels was investigated, by measuring the contact angle of water on the hydrogel surfaces. The morphology and metabolism of the HT29 cells was also investigated on the six different hydrogels. A 5o/ gelatin hydrogel was selected for the formation of lumens inside the LOC devices because it was the most hydrophilic hydrogel surface. HT29 cells formed a confluent monolayer on the hydrogel after 72 hours of incubation. The cells also adhered inside the lumens, but did not form a confluent 2D monolayer, possibly due to the initial seeding concentration being too low. The LOC device provides a template from which future work could focus on the optimisation of cell culture inside the lumen.
Due to the production of a toxin (Bacteroides fragilis toxin (EFT)), the carriage of enterotoxigenic B. fragilis (ETEF) in the colonic mucosa of humans is associated with an increased risk of colon carcinogenesis. EFT is a metalloprotease, which has been associated with the cleavage of E-cadherin adherens junctions in the intercellular space. Once cleaved, the gut epithelium becomes compromised, increasing the risk of carcinogenesis. To investigate the role of the EFT as a driver of colon carcinogenesis. EFT-treated HT29 cells grown on 5o/ gelatin hydrogels were exposed to EFT for 12 or 24 hours. These experiments were conducted on cells in fluorodishes and demonstrated the loss of E-cadherin and cell rounding after 12 and 24 hours. In future, these experiments should be carried out on confluent cells lining the lumen of the chip, as the work carried out in this study has provided the basis for this to be executed.