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Mycobacterial Infections of Macrophages in 3D Matrices

Ramberg, Oda Helgesen
Master thesis
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URI
http://hdl.handle.net/11250/2615497
Date
2015
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  • Institutt for bioteknologi og matvitenskap [900]
Abstract
This work highlights a novel approach on studying mycobacterial infection moving from traditional 2D cultures to 3D environments. The mechanisms underlying infection by mycobacteria, and the abilities of the bacteria to survive phagocytosis by macrophages, are not fully known. To improve the biological relevance of in vitro infection studies, alginate hydrogels and Matrigel were tested as 3D scaffolds for cell growth and mycobacterial infections, with macrophages of different origins and DsRed-expressing Mycobacterium avium (Mav). The main objective of this study was to evaluate the ability to use 3D hydrogels as extracellular matrix (ECM) mimics for cell growth and infection studies, based on cell viability, morphology, adhesion, migration and progress in infection. Two strategies for infection in 3D were assessed; immobilization of cells together with Mav, and seeding of cells on top of gels containing Mav. Confocal microscopy was used to analyze cells and Mav inside the hydrogels.

Matrigel was successfully shown to be an effective ECM mimic for cell growth and infection studies. Macrophages immobilized and seeded on top of Matrigel containing Mav were found to utilize the 3D environments for adhesion and migration in the process of bacterial phagocytosis, and infection was found to increase over time in terms of number of infected cells and number of intracellular Mav. Based on the elongated morphology of the cells, seeding of cells on top of gels was concluded to be the most preferable approach, compared to immobilization. Functionalization of alginates with bioactive peptides was not found to improve cell viability of immobilized macrophages, and though infected immobilized cells were found in all alginates, the infection did not increase over time. However, the cells were able to utilize available peptides for adhesion and migration from the top of functionalized gels. Migrated cells adopted an elongated morphology, and were infected by Mav inside the matrices. By comparing the viability and morphology of cell line RAW 264.7 to primary macrophages from both murine and human origin, the primary cells proved most successful in 3D cultures, which also strengthen the clinical relevance of the study.

The findings in this study can be used for further work on mycobacterial infection and cell migration studies to gain new information on cell functions in 3D tissues.
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NTNU

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