Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.766550
Title: The development of in vitro models of human salivary glands
Author: A Rahman, Zulaiha
ISNI:       0000 0004 7655 3752
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2018
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Abstract:
Introduction: At present, due to the lack of good, representative, human models, it is not possible to fully investigate the development of a number of salivary gland disorders such as tumour development, Sjogren's syndrome or viral-associated infections. Radiotherapy for head and neck cancer, and chemotherapy for non-oral cancers, can also lead to salivary gland damage, reduced salivary flow, xerostomia and a worst-case scenario of severe, debilitating mucositis with potentially lifethreatening systemic infections. An artificial salivary gland is a novel alternative treatment for patients who suffer from diseased or damaged glands and subsequent hyposalivation which often leads to untreatable and irreversible xerostomia. Salivary gland (SG) organoids developed from mice SG cells exhibit self-organizing properties with structural and functional properties closely resembling the native organ. Thus, organoid technology has potential as a model of human organ development, for the investigation of human pathologies in vitro and also for regenerative medicine. Aims: To develop in vitro three-dimensional culture system of human salivary glands, to elucidate the pathogenesis of salivary gland disease and to further understand the development of salivary glands for regenerative studies. Methods: Organoids were developed from biopsy samples of normal human sublingual gland tissue. Cells were isolated and cultured in extracellular matrices (Matrigel and/or Myogel), as well as on Polymerised High Internal Phase Emulsions (PolyHIPES) scaffolds. They were cultured either at an Air Liquid Interface (ALI) or were submerged in tissue culture medium (non-ALI) and Wnt-3A, R-spondin1, EGF, and FGF2 were added to supplement the medium. TGFß, BMP, and LIMK inhibitors were added to an enriched media for further differentiation studies. SG organoids were infected with Staphylococcus aureus in preliminary study of glandular infection. Haematoxylin and eosin stained sections of the cultures were used to visualise growth. RT-PCR, immunohistochemistry and immunofluorescence were used to determine the differential expression of cell specific markers. Results: Overall, the data from this study indicate that salivary gland cells from single cell suspensions were able to proliferate and differentiate to form small structures resembling mini-glands (organoids) for up to 14-days in a defined culture system. Organoid structures developed when the cells were grown in Matrigel but this was less obvious when grown in Myogel. Compared to those grown at an ALI, non-ALI organoids were significantly fewer in number and smaller in size. Organoids cultured in Matrigel at ALI formed buds and branches, and expressed acinar cellspecific markers (amylase, AQP5 and MUC7), as well as staining positively for CK5. Inhibition of TGFß, BMP and LIMK signalling promoted cell proliferation and induced cell differentiation. Organoids cultured in the presence of the TGFß inhibitor, A8301, and in the presence of all three inhibitors, ALL-media, displayed distinct characteristics that closely mimicked native glands and expressed CK5, BPIFA2, AQP5, CK7 and E-cadherin, whilst those cultured in the presence of the BMP inhibitor, DMH1, demonstrated the growth of duct-like structures. a-AMY activity was significantly higher in SG organoids grown in the presence of the LIMK inhibitor, SR7826. Finally, cells grown in PolyHIPE scaffolds were able to form budding and branching structures more closely resembling human salivary glands Conclusion: Our culture system enables the growth of human salivary gland organoids. This culture system can be used to study the initial stages of infectious disease and the pathogenesis of other salivary gland diseases. This study provides a competent in vitro culture system that can be used in further study on human SG development, pathophysiology as well as pioneering a novel pathway in salivary gland regeneration.
Supervisor: Bingle, Lynne ; Bingle, Colin Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.766550  DOI: Not available
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