Abstract
Standard cell culture models have been used to investigate disease pathology and to test new therapies for over fifty years. However, these model systems have often failed to mimic the changes occurring within three-dimensional (3-D) space where pathology occurs in vivo. To truthfully represent this, an emerging paradigm in biology is the importance of modelling disease in a physiologically relevant 3-D environment. One of the approaches for 3-D cell culture is bioelectrospray technology. This technique uses an alginate-based 3-D environment as an inert backbone within which mammalian cells and extracellular matrix can be incorporated. These alginate-based matrices produce highly reproducible results and can be mixed with different extracellular matrix components. This protocol describes a 3-D system incorporating mycobacteria, primary human blood mononuclear cells and collagen-alginate matrix to dissect the host-pathogen interaction in tuberculosis.
Highlights
[Background] Mycobacterium tuberculosis (Mtb) is a pathogen of global public health importance that causes a mortality of 1.8 million people per year and morbidity of 10 million worldwide (WHO, 2016)
One of challenges to the tuberculosis (TB) field is the availability of model systems to interrogate the host-pathogen interaction, as widely used animal models do not fully reflect pathology in humans
Mtb is an obligate pathogen of man and so we hypothesized that a model system requires human cells, virulent mycobacteria, 3-dimensional organization, extracellular matrix, longitudinal readouts and the ability to modulate the environment over time
Summary
Note: Sterilize lab tissue paper by autoclaving and keep it sterile until use. Methylated spirit (Fisher Scientific, catalog number: 11492874) 15. RPMI 1640 medium (Thermo Fisher Scientific, GibcoTM, catalog number: A1049101) 16. EDTA powder (Fisher Scientific, catalog number: 10522965) 17. Versene solution (Thermo Fisher Scientific, GibcoTM, catalog number: 15040033) 18. Hank’s balanced salt solution (HBSS), no calcium, no magnesium (Thermo Fisher Scientific, GibcoTM, catalog number: 14170112) 19. HBSS, calcium, magnesium (Thermo Fisher Scientific, GibcoTM, catalog number: 24020117) 20. 1 N NaOH cell culture grade solution (Sigma-Aldrich, catalog number: S2770-100ML) 24. 1 M HEPES (Thermo Fisher Scientific, GibcoTM, catalog number: 15630106) 25. 7.5% NaHCO3 solution (Thermo Fisher Scientific, catalog number: 25080060) 26. 96%, extra pure, powder, anhydrous, ACROS OrganicsTM (Thermo Fisher Scientific, catalog number: 10021681) Manufacturer: Acros Organics, catalog number: 349615000.
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