A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells
July 5th, 2018
We have developed a multi-well format polyacrylamide-based assay for probing the effect of extracellular matrix stiffness on bacterial infection of adherent cells. This assay is compatible with flow cytometry, immunostaining, and traction force microscopy, allowing for quantitative measurements of the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria.
Overview
This article presents a quantitative methodology to investigate how extracellular matrix stiffness influences the susceptibility of adherent endothelial cells to bacterial infection, specifically using Listeria monocytogenes as a model pathogen. The protocol enables high-throughput screening of multiple conditions, combining infection assays with biomechanical analysis to elucidate the interplay between matrix mechanics and host-pathogen interactions.
Key Study Components
Area of Science
- Cellular microbiology
- Mechanobiology
- Host-pathogen interactions
- Cell biomechanics
Background
- Extracellular matrix stiffness is a key mechanical cue affecting cell behavior and fate.
- The impact of matrix stiffness on host cell susceptibility to bacterial infection is not well understood.
- Bacterial infection may alter the biomechanics of host cells.
- Understanding these interactions is critical for the emerging field of host-pathogen biomechanics.
Purpose of Study
- To determine whether the stiffness of the extracellular matrix affects the susceptibility of endothelial cells to L. monocytogenes infection.
- To assess how bacterial infection alters the biomechanics of host endothelial cells.
- To develop a robust, multi-well assay for parallel analysis of these phenomena.
Methods Used
- Preparation of polyacrylamide hydrogels with tunable stiffness in 24-well plates.
- Surface functionalization and collagen coating of hydrogels for cell adhesion.
- Seeding of human microvascular endothelial cells (HMEC-1) onto hydrogels.
- Infection of cells with L. monocytogenes at defined multiplicities of infection (MOI).
- Synchronization of infection via centrifugation and subsequent incubation.
- Quantification of infection using flow cytometry and immunostaining followed by microscopy.
- Live-cell imaging to monitor bacterial spread.
- Atomic force microscopy (AFM) to confirm hydrogel stiffness and assess cell biomechanics.
Main Results
- Flow cytometry revealed that infection rates of HMEC-1 cells by L. monocytogenes were approximately two-fold higher on stiff (70 kPa) versus soft (0.6 kPa) hydrogels.
- Immunostaining showed significantly more bacteria adhered to and internalized by cells on stiff matrices compared to soft ones.
- Live-cell imaging enabled visualization of bacterial spread across monolayers on different stiffness substrates.
- AFM confirmed the precise stiffness of the hydrogels and can be used to assess changes in host cell mechanics post-infection.
Conclusions
- Matrix stiffness significantly modulates the susceptibility of endothelial cells to bacterial infection.
- Bacterial infection can alter the biomechanics of host cells, and this effect may depend on matrix stiffness.
- The described multi-well assay is broadly applicable for studies of cell biomechanics and mechanobiology in the context of infection and beyond.
Frequently Asked Questions
Executive Industry Relevance
Quantitative assessment of extracellular matrix stiffness on bacterial infection susceptibility addresses a critical gap in host-pathogen mechanobiology. This multi-well assay enables high-throughput, reproducible evaluation of biomechanical factors influencing infection, supporting predictive confidence in early discovery and target validation. The approach enhances portfolio decision-making by clarifying mechanistic drivers of infection risk in disease-relevant systems.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of how mechanical microenvironment modulates host cell susceptibility to infection.
- Supports biological de-risking by quantifying infection outcomes across defined matrix stiffness conditions.
- Facilitates functional target validation by linking extracellular cues to infection phenotypes.
- Improves predictive confidence for prioritizing mechanistic hypotheses in host-pathogen research.
Screening & Assay Development
- Prepares validated, tunable-stiffness biological systems for downstream infection and biomechanics assays.
- Standardizes infection quantification using flow cytometry and microscopy for reproducible outputs.
- Enables parallel screening of multiple conditions, increasing throughput and scalability.
- Supports reliable evaluation of compound or genetic perturbations in a controlled mechanical context.
Translational & Preclinical Research
- Aligns in vitro infection models with tissue-relevant mechanical properties for translational continuity.
- Provides a platform for assessing biomechanical biomarkers of infection susceptibility.
- Facilitates risk-adjusted advancement by clarifying matrix-dependent infection mechanisms.
- Enables mechanistic de-risking for preclinical model selection and validation.
Pipeline & Workflow Integration
This assay bridges early discovery and preclinical research by enabling hypothesis-driven testing of matrix mechanics on infection outcomes, supporting lead identification and mechanistic de-risking.
- Discovery Biology: Quantifies the impact of extracellular stiffness on host-pathogen interactions, clarifying biological pathways.
- Screening: Delivers standardized, quantitative infection readouts suitable for comparative analysis.
- Analytics: Integrates flow cytometry, microscopy, and AFM for robust measurement of infection and biomechanics.
- Translational Research: Models disease-relevant mechanical environments to inform preclinical study design.
- Enterprise Reuse: Provides a reusable, scalable platform adaptable to diverse cell types and pathogens.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in infection susceptibility studies.
- Operational Value: Standardizes workflows for reproducibility and parallelization across multiple conditions.
- Strategic Value: Informs go/no-go decisions by linking mechanical cues to infection risk.
- Portfolio Impact: Enables risk-adjusted prioritization of targets and models based on biomechanical insights.
Implementation Considerations
- Requires expertise in cell culture, infection assays, and biomechanical analysis.
- Needs access to flow cytometry, fluorescence microscopy, and atomic force microscopy instrumentation.
- Demands rigorous cross-team standardization for hydrogel preparation and infection quantification.
- Adaptable to various adherent cell types and bacterial pathogens with protocol optimization.
- Strict biosafety and contamination control are essential when handling pathogenic bacteria.
Biopharma FAQs
Chapters in this video
0:05
Title
0:39
Manufacturing Thin Two-layered Polyacrylamide Hydrogels on Multi-well Plates
1:36
Polyacrylamide Hydrogel Fabrication
5:22
Infection of Human Microvascular Endothelial Cells with L. monocytogenes
7:11
Flow Cytometry to Quantify Extracellular-matrix-stiffness Dependent Susceptibility of Host Cells to Infection
8:02
Quantitative Time-lapse Microscopy to Assess Extracellular-matrix-stiffness Dependent L. monocytogenes Dissemination Through Endothelial Cells
9:01
Results: The Effect of Extracellular Matrix Stiffness on L. monocytogenes Infection of HMEC-1 Cells
10:32
Conclusion
