Long-term, live cell analysis of host-pathogen interactions using the CellASIC® ONIX System

Introduction

Host-pathogen interactions represent a signifcant area of biomedical research, encompassing the study of how viruses, bacteria, and other organisms cause infections and diseases or alter normal physiology of host cells and tissues1. As the feld of cellular systems biology advances, there is increasing interest in using in vitro cell culture models to study host-pathogen interactions2.

The proper study of host-pathogen interactions requires careful control of the cell environment to simulate physiologic conditions. An in vitro model that can replicate infection parameters, including flow rate, exposure time, solution type, and timed introduction of therapeutic agents while sustaining long-term live cell analysis enables experiments that are diffcult or impossible with standard methods. In many cases, it is crucial to precisely manipulate the exposure of pathogen to the cultured host cells: too low an exposure may prevent appropriate interactions, while too high an exposure could cause confounding effects (such as bacterial overgrowth and nutrient starvation) independent of the desired host-pathogen interaction. In addition, information on the effect of exposure times and flow conditions could prove valuable for understanding the mechanisms of disease for many infectious agents. A further challenge in host-pathogen research is the ability to sustain infections in vitro, since cultured host cells often de-differentiate in culture and no longer support pathogen responses found in vivo. For certain pathogens, the time scale of infection even under optimal conditions could take days or weeks, placing additional burdens on the cell culture method. Recent advances in microfluidics-based cell culture technologies offer potential solutions to these problems3.

The CellASIC® ONIX Microfluidic System is well suited for host-pathogen studies by providing a stable, longterm culture environment for host cells (including primary cells) with controlled pathogen exposure. The continuous perfusion format and the ability to switch media solutions enable wash-out of pathogens from the chamber and subsequent monitoring of host cell response over many days. The enclosed small volume of the culture chamber also provides practical advantages for working with infectious agents during live cell analysis. The design of the microfluidic plates enables the dynamics of infection to be tracked using standard inverted microscopes.

Here, we demonstrate a host-pathogen experiment using human intestinal cells infected with engineered E. coli strains (Figure 1). Both an invasive and noninvasive bacterial strain were monitored for long-term infection with time-lapsed analysis up to 24 hours in the CellASIC® M04S microfluidic plate.

Figure 1. Human colon adenocarcinoma cells (HT-29) cultured in the M04S microfluidic chamber and infected with an invasive strain of E. coli. (A) Phase contrast image showing apoptotic cells, and (B) fluorescence image showing invaded bacteria (red) and cell viability (green). Images were acquired with a 100X objective lens.

Plate design

The M04S microfluidic plate is built on the CellASIC® ONIX Microfluidic Platform. The plate has a standard 96-well footprint to ft typical microscope stage holders. The custom well layout was designed to maximize live cell analysis capabilities. The M04S plate has four independent units (A-D), with each unit containing eight wells (one gravity inlet, four switching inlets, one cell inlet, two waste outlets). The four cell culture chambers are centralized under a single large viewing window (Figure 2). The chamber-to- chamber distance is 5.2 mm, reducing objective travel time and focus drift. The bottom surface of the plate is a #1.5 thickness (170 µm) optical glass slide to maximize quality of high resolution, high numerical aperture viewing. The plate houses all experiment solutions allowing control with an external pneumatic manifold (Figure 3). The manifold lets the user direct flow rates and select exposure solutions without perturbing the microscope stage. The programmable software interface automates flow switching times. A gas line allows control of the environment within the microchambers through a network of gas-permeable air diffusion channels. Temperature is regulated through an on-board heater/chiller on the manifold.

Figure 2. The M04S plate contains four independent flow units (A-D), each with four upstream solution inlets, a gravity flow inlet,a cell inlet, and two waste wells. The culture chamber is 2.8 mm in diameter (120 µm height) and is surrounded with a microfabricated perfusion barrier (4 µm pores). Inlet 1 is a gravity flow well, allowing long term cell culture in a standard incubator without a pressure system. Continuous flow of solutions from the inlets creates a dynamic exposure profle during live cell analysis.

Figure 3. Side view schematic of the microfluidic plate with microincubation manifold on a microscope stage. The bottom surface of the microfluidic plate is a thin glass sheet, allowing high quality cell viewing. The plate is sealed to a pneumatic manifold, allowing user control of the flow profle during analysis. Additional air channels allow control of the gas environment.

Bacterial infection model

To demonstrate time-lapsed analysis of host-pathogen interactions in the microfluidic culture system, a human colon adenocarcinoma cell line (HT-29, ATCC®) was exposed to two E. coli strains (courtesy of Tim Lu, MIT). The strains tested included one capable of HT-29 infection and one that could not. A schematic of how the perfusion system was used to monitor invasion is depicted in Figure 4.

Figure 4. Schematic of host-pathogen experiment. (A) Human cells are cultured in the M04S microfluidic plate to establish a healthy, differentiated, and stable host population under continuous medium perfusion. (B) Pathogens (bacteria) are introduced by flow to expose the host cells. (C) Medium perfusion is re-established, allowing long-term monitoring of host-pathogen interaction.

Figure 5. Time-lapsed analysis of bacterial invasion into human HT-29 cells. A) Phase contrast and (B) fluorescence images of an invasive strain of E. coli. (C) Phase contrast and (D) fluorescence images of a non-invasive E. coli strain. Both strains are designed to express mCherry following invasion into HT-29 cells.

The experiment was prepared by culturing HT-29 cells in the M04S microfluidic plate in a standard cell culture incubator for three days with gravity perfusion of McCoy’s 5A medium + 10% fetal bovine serum (FBS). This allowed the cells to form close cell-cell contacts and enter growth phase. On the day of the experiment, E. coli cells were cultured in Luria Broth (LB) on a shaker to achieve log phase growth. The M04S plate was then loaded with culture medium (McCoy’s 5A + 10% FBS) in well 2 and bacterial suspension in well 3. Within each plate, the invasive strain, non-invasive strain, and no bacteria control were run in parallel chambers. For live cell analysis, the M04S plate was sealed to the CellASIC® ONIX Microincubator Controller manifold (for perfusion, temperature, and gas control) and viewed using an Olympus IX71 microscope. Conditions were set at 37°C and 5% CO2 for the duration of the experiment. The cells were initially perfused with culture medium for 1 hour at a flow rate of 5 µL/hr, and then exposed to the bacteria solution for 30 minutes, followed by wash-out by medium flow at 5 µL/hr for the remainder of the experiment. Figure 5 shows images comparing the invasive strain (a and b) against the non-invasive strain (c and d). During bacterial exposure, there was a high abundance of both strains visible in the solution (t = 0). The subsequent wash-out removed the majority of the bacteria, allowing the HT-29 cells to continue healthy culture. In the non-invasive condition, the HT-29 cells were able to continue growth and exhibit a healthy morphology. In the invasive strain case, the HT-29 cells showed clear signs of stress by 6 hours, with widespread cell death occurring by 12 hours. The fluorescence channels showed mCherry expression by the bacterial strains triggered by invasion, verifying the cell death as a result of invasion instead of nutrient competition.

Figure 6 shows higher magnifcation images of the HT-29 cells exposed to the invasive and non-invasive strains. Here, the cells were stained with the green fluorescent Calcein AM viability marker, and the bacteria were red fluorescent. In the invasive case, there was clear co-localization of the bacteria with the human cells, whereas in the non-invasive case, the bacteria were effectively excluded from the HT-29 colony. When tracked over time, the invasive bacteria triggered apoptosis and eventually cell rupture, promoting bacterial growth and continued invasion. By 15 hours after initial exposure, almost all of the HT-29 cells in the invasive chamber had died, while the majority of the HT-29 cells in the non-invasive chamber remained viable.

Figure 6. Images of (A) an invasive strain and (B) a non-invasive strain of E. coli after exposure to human HT-29 cells, washout, and perfusion culture. Bacteria expressed mCherry, and HT-29 cells stained with Calcein AM. Panel (A) was acquired with a 100X objective lens, and panel (B) was acquired with a 60X objective lens.

The continuous perfusion culture format was highly benefcial in promoting long-term host-pathogen response. Without the presence of the wash-out flow, the bacteria quickly overcrowded the culture and led to cell death even in the absence of invasion. In addition, the low chamber height (120 µm) improved the ability to focus on bacterial cells in suspension. While not investigated in detail in this study, the flow environment also appeared to play a role in how the pathogen accesses host cells. For example, cells at the periphery of a colony were much more susceptible to invasion, as well as areas that served as “harbors” and prevented pathogen wash-out. It was also observed that the flow rate could play a role in the effectiveness of cell invasion.

Summary

The CellASIC® ONIX Microfluidic System provides a number of important features for live cell analysis of host-pathogen interactions. The ability to precisely control the cell culture environment on the microscope stage for long periods of time (hours to days) while maintaining a steady state perfusion of medium gives the researcher unprecedented access to tracking host-pathogen dynamics. The M04S microfluidic plate is well suited for the culture of a large variety of adherent host cells, and the programmable solution switching capability allows controlled exposure of pathogens, including bacteria, viruses, and other particles. The ease of use and flexibility of this experiment platform facilitate its potential applications in a wide range of live cell studies.

References

  1. Casadevall A, Pirofski LA. Host-pathogen interactions: basic concepts of microbial commensalism, colonization, infection, and disease. Infect Immun. 2000 Dec;68(12):6511-8.
  2. Hoppe AD, Seveau S, Swanson JA. Live cell fluorescence microscopy to study microbial pathogenesis. Cell Microbiol. 2009 Apr;11(4):540-50.
  3. Lee P, Gaige T, Hung P. Microfluidic systems for live cell imaging. Methods Cell Biol. 2011;102:77-103.

E. coli strains courtesy of Professor Tim Lu of the Massachusetts Institute of Technology.

 

     
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