Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

Summary

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

Abstract

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

Protocol

1. Fabrication of silicon masters using standard SU-8 photolithography¹ (not shown in this video).

1. Use standard SU-8 photolithography methods to create a SU-8 "masters" (SU-8 2050, MicroChem, Newton, MA) for fabricating the different components (PDMS membranes of different thickness, co-culture chamber) in a cleanroom. Such master molds can be fabricated at any microfabrication facility (e.g., Stanford Microfluidics Foundry; http://thebigone.stanford.edu/foundry/).
2. Prior to PDMS replication, expose the SU-8 masters to fluorosilane vapor in a desiccator attached t....

Discussion

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 impossib.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
SU-8 2050	MicroChem Corp.
high-resolution (16,256 dpi) photolithography mask	Fineline-Imaging Inc, CO
Trichloro(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)silane	Sigma-Aldrich	77279
PDMS	Dow Corning	184 SIL ELAST KIT 0.5KG
DMEM	Thermo Fisher Scientific, Inc.	SH30002.02
Programmable spin coater	Laurell Tech Corp	WS0650S
Mask aligner	Neurotronix	Q4000
Oxygen plasma etcher	March Plasma System, CA	CS-1701
Syringe pump	Harvard Apparatus
Live/Dead Viability/Cytotoxicity Kit	Invitrogen	L-3224