1McFerrin Department of Chemical Engineering, Texas A&M University, 2Department of Biomedical Engineering, Texas A&M University
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Kim, J., Hegde, M., Jayaraman, A. Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions. J. Vis. Exp. (38), e1749, doi:10.3791/1749 (2010).
1. Fabrication of silicon masters using standard SU-8 photolithography1 (not shown in this video).
2. Replica molding of PDMS from SU-8 master2
3. Bonding of Multilayer PDMS devices: This device is assembled with three layers -a top layer which contains the main channel, a middle pneumatic layer, and a bottom bacterial layer that contains the channels for bacteria. The three layers are assembled as described below.
4. Assembling the PDMS device3,4
5. Treating the glass surface with fibronectin
6. Formation and measurement of commensal E. coli biofilm in islands
7. Seeding eukaryotic cells around bacterial islands6-8
8. Introduction of pathogenic bacteria into the island and exposure to eukaryotic cells
9. Representative Results 9
The sequence of events in GI tract infections was mimicked by developing a monolayer of HeLa cells and a commensal E. coli biofilm and exposing EHEC to the commensal biofilm prior to infection of HeLa cells. The steps involved in fabrication of the microfluidic co-culture model are shown in Figure 1. Figure 2A shows a 3-dimensional rendering of the co-culture device. The two regions in the co-culture device the eukaryotic cell culture chamber and the bacterial island are shown in Figure 2B with different color dyes (purple for the eukaryotic regions and green for the bacterial island). The feasibility of isolating the bacterial culture regions from the surrounding eukaryotic region is shown in Figure 2C using color dyes. Figure 3 shows representative results from co-culture of epithelial cells and bacteria. Figure 3A shows colonization of the commensal biofilm (green) island by EHEC (red). Figure 3B shows the juxtaposition of epithelial cells and commensal E. coli with EHEC in the bacterial island. The RFP expressing EHEC and GFP expressing commensal bacteria are localized in the island when the PDMS wall is lowered, illustrating the feasibility of isolating the bacterial island from epithelial cells.
Figure 1: Cell seeding scheme in the co-culture model. (1) The PDMS wall is lowered to form an island, commensal bacteria are introduced into the island, and fibronectin is flowed around the island. (2) HeLa cells are seeded in the regions surrounding the island. (3) After HeLa cells reach confluence and the commensal biofilm has developed, EHEC is introduced into the island. (4) The PDMS wall is lifted up to expose HeLa cells surrounding the island to EHEC. Inset shows details of valve operation.
Figure 2: Microfluidic model for co-culture of epithelial cells and bacteria. (A) Three-dimensional rendition of the co-culture device showing pneumatically-actuated trapping regions for forming bacterial islands among epithelial cells. Each bacterial island (1200 μm diameter and 1000 μm apart) has a separate inlet and outlet for providing growth media and removing waste from the island. (B) Micrograph of the co-culture device with color dyes showing the different regions (epithelial cell zone, bacterial islands). (C) The fidelity of the pneumatic trapping system is shown by lowering the PDMS wall (left panel) using a pneumatically-activated channel (blue), introducing purple dye into the closed island islands, and flowing yellow dye around it for 48 h. When the PDMS wall is raised (right panel), the island region is exposed to the surrounding yellow dye. Scale bar represents 500 μm.
Figure 3: Co-culture of HeLa cells and bacteria. (A) Fluorescence image of RFP-expressing EHEC and GFP-expressing E. coli BW25113 in island. (B) Overlay of transmitted, green, and red fluorescence images in the device. Scale bar represents 200 μm.
Conventional assays for pathogen attachment and colonization utilize a monolayer of eukaryotic cells in tissue culture plates into which pathogens are added. These models are not physiologically relevant as they do not incorporate a commensal bacterial biofilm developed on eukaryotic cells. Simple addition of a pre-grown bacterial culture to eukaryotic cells is unlikely to lead to this conformation as biofilms are highly organized structures that develop over time, and it is extremely difficult, if not virtually impossible, to culture eukaryotic cells in the presence of bacteria for extended periods of time without significant loss in viability. Since pathogens do not navigate through a commensal biofilm in these models to attach to epithelial cells, these models do not accurately mimic the organization of epithelial cells and commensal bacteria in the GI tract. Here, we outline the development of a microfluidic device that uses pneumatically-controlled trapping for co-culture of eukaryotic cells and bacteria. Using HeLa cells as the model eukaryotic cell line and EHEC as the model pathogen, we show that the co-culture device can keep the island region isolated as well as support the cultivation and development of a HeLa cell monolayer and a commensal E. coli biofilm. The microfluidic co-culture model not only enables localization of commensal bacteria and epithelial cells, but also pre-exposes pathogens to commensal bacteria prior to encountering epithelial cells; thereby, presenting a more physiologically relevant environment during colonization. In addition, the co-culture model can be a useful tool for fundamental studies focused on investigating the role of specific signals on EHEC infectivity as well as for applications such as screening potential probiotic strains.
This work was supported in part by the National Science Foundation (CBET 0846453) and the National Institutes of Health (1R01GM089999).
|SU-8 2050||MicroChem Corp.|
|high-resolution (16,256 dpi) photolithography mask||Fineline-Imaging Inc, CO|
|PDMS||Dow Corning||184 SIL ELAST KIT 0.5KG|
|DMEM||Thermo Fisher Scientific, Inc.||SH30002.02|
|Programmable spin coater||Laurell Tech Corp||WS0650S|
|Oxygen plasma etcher||March Plasma System, CA||CS-1701|
|Syringe pump||Harvard Apparatus|
|Live/Dead Viability/Cytotoxicity Kit||Invitrogen||L-3224|