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Investigation of in vitro air-liquid interface (ALI) models of lung epithelium
Malmö University, Faculty of Health and Society (HS), Department of Biomedical Science (BMV).
2021 (English)Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
Abstract [en]

The rapid spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) originated from Wuhan, China, in December 2019, led to a surge in scientific research of the virus and its host receptor angiotensin-converting enzyme-2 (ACE-2). The viral entry depends on the proteolytic cleavage by transmembrane protease serine 2 (TMPRSS2) and a membrane sheddase protease called ADAM17,  which is involved in the release of the membrane-bound ACE2. Since surface-bound ACE2 is expressed on many different cell types, we included lung cells, kidney cells, and a breast cancer cell line in this study. In general, 2D models of cell cultures are most commonly used but do not consider the biological 3D environment of the cells. To develop more physiological in vitro models, and to better mimic the in vivo environment, air-liquid interface (ALI) lung cell cultures have been developed. This study aims to establish an ALI of lung epithelial cells as a future model to investigate the interaction between the receptor-binding domain (RBD) and of the spike protein of SARS-CoV-2 to ACE2 TMPRSS2 and ADAM17. Moreover, the mRNA and protein surface expression of ACE2, TMPRSS2, and ADAM17 was compared to monolayer cultured cells. Our results show that monolayer cultured kidney and breast cells expressed higher levels of ACE2 compared to lung epithelial cells. The human lung adenocarcinoma cell line Calu-3 showed a well-differentiated pseudostratified layer after three weeks in ALI culture, including mucus production as shown by PAS staining. Moreover, after RBD and RBD+anti-FC treatment, the cellular response of the ALI cultures showed an opposite gene expression of  IL-6, TNFα, and ADAM17 compared to monolayer cultured Calu-3 cells. Furthermore, the soluble form of ACE2 was shown to be detected in larger amounts in ALI of Calu-3 cells compared to monolayer cultured cells. We conclude that the Calu-3 cells cultured at the ALI generated a morphological representative model of the airway epithelium and may serve as an in vitro model to investigate responses of host cells encountering viruses in future studies.

Place, publisher, year, edition, pages
2021. , p. 27
Keywords [en]
ACE2, ADAM17, Air-Liquid interface, Calu-3, SARS-CoV-2
National Category
Cell Biology Immunology
Identifiers
URN: urn:nbn:se:mau:diva-43593OAI: oai:DiVA.org:mau-43593DiVA, id: diva2:1567246
Educational program
HS Biomedical Surface Science
Supervisors
Examiners
Available from: 2021-08-10 Created: 2021-06-16 Last updated: 2021-08-10Bibliographically approved

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