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. 2017 Jul 20;7(14):e2418.
doi: 10.21769/BioProtoc.2418.

Bioelectrospray Methodology for Dissection of the Host-pathogen Interaction in Human Tuberculosis

Liku B Tezera  1 , Magdalena K Bielecka  1 , Paul T Elkington  1
Affiliations

Bioelectrospray Methodology for Dissection of the Host-pathogen Interaction in Human Tuberculosis

Liku B Tezera et al. Bio Protoc. .

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: Alginate-based matrices; Bioelectrospray; Collagen; Extracellular matrix; Multicellular 3-D cell culture; Tuberculosis.

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Figures

Figure 1.
Figure 1.. Preparation of cells for encapsulation.
Cells are recovered from a 75 cm2 flask and pelleted in a Falcon by centrifugation, and then mixed with alginate-collagen matrix in a 7 ml bijou container (usually 25 million cells/5ml of matrix mix). Also see Video 1.
Video 1.
Video 1.. Mixing alginate with PBMCs prior to bioelectrospraying
Figure 2.
Figure 2.. Nisco electrostatic encapsulator with washed and alcohol sterilized arm, sterile nozzle, sterile silicon tubes and crystalizing glass beakers.
1. Nozzle (0.7 φ) attached to the nozzle holder; 2. Electrostatic accelerator arm for the electrostatic bead generator VARv1; 3. Electrode cable; 4. Silicon tubes with connector attached at the end; 5. Borosilicate crystalizing glass beakers with spout with magnetic stirrers (1 cm); 6. Stirrer; 7. Ring on the electrostatic accelerator arm; 8. Ruler for setting correct needle height.
Figure 3.
Figure 3.. Biolelectrospraying microspheres.
Matrix in syringe is injected to the bioelectrosprying machine and microspheres are formed. A syringe filled with matrix was set up on syringe pump (A) so that it will inject the matrix into silicon tube (B) connected to the electrostatic bead generator. The syringe driver is sitting on jack (C) for height adjustment. E. Unused crystalizing glass beakers on the roof of the bioelectrospray machine; F. Housing with doors to enclose the electrostatic bead generator; G. High-voltage switch on/off (white, on/green, off) which is left of potentiometers for optional peristaltic pump, agitator speed and voltage on electrode. Voltage indicator displaying 7.0 kV. Biobin (H) and old media bottle (I) containing surfanios (10%) for discarding biohazardous waste.
Figure 4.
Figure 4.. Microspheres are formed in HBSS solution with 100 mM CaCl2.
A mix of collagen-alginate with cells in 2 mm diameter silicon tube at a specific rate and microspheres are formed in the gelling bath. Also see Video 3.
Video 2.
Video 2.. Setting up the bioelectrospray system
Video 3.
Video 3.. Bioelectrospray system in operation with microspheres being formed
Figure 5.
Figure 5.. Microspheres in HBSS with Ca/Mg after transferred from the gelling bath.
Also see Video 4.
Video 4.
Video 4.. Decanting microspheres after generation
See this image and copyright information in PMC

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References

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