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.
Keywords: Bioelectrospray, Alginate-based matrices, Multicellular 3-D cell culture, Tuberculosis, Collagen, Extracellular matrix
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). Despite substantial investment in research, much greater understanding of the host-pathogen interaction is required to improve prevention and treatment. Currently, the pathogen is becoming increasingly resistant to commonly used drugs, with the emergence of extensively drug-resistant Mtb. 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. Hence, there is an urgent need to complement these animal models by developing a physiologically relevant in vitro environment (Bielecka et al., 2017; Tezera et al., 2017). 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.This protocol describes a physiologically relevant in vitro environment by utilizing human cells, extracellular matrix components and live Mtb using a bioelectrospray model to mimic human Mtb infection. This 3-D model is different from other models by using an extracellular matrix that can be released by de-capsulation so that downstream analysis of cells within the matrix can be performed. Furthermore, this methodology has wide potential applicability to investigate infectious, inflammatory and neoplastic diseases and develop novel drug regimens and vaccination approaches.
Materials and Reagents
Equipment
Procedure
Data analysis
Once the microspheres are formed, one can consider them as an individual’s cells/collection of cells as the microspheres are permeable, like sponges. Microspheres can be set up in 96-well plates for assays testing viability, necrosis and apoptosis. Mycobacterial growth can be measured by luminescence if the bacterium has a luminescence reporter plasmid, or directly by counting colony forming units on Middlebrook 7H11 agar after de-capsulation. Cellular composition can be analyzed by flow cytometry after de-capsulation and paraformaldehyde fixation, and RNA analysis by de-capsulation and cellular lysis with Trizol. Data is analyzed according to standard workflows, our routine is a minimul of 2 separate donors with each experimental variable in triplicate.
Recipes
Acknowledgments
We would like to thank S. N. Jayasinghe from University College London, United Kingdom for all the technical support and advice on the bioelectrospray technology. This work is funded by the grant from the US National Institute for Health R33AI102239, the UK National Centre for the 3Rs NC/L001039/1 and the Antimicrobial Resistance Cross Council Initiative supported by the seven research councils MR/N006631/1.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.