Immune System Models
Tunable In Vitro Assays for Immune Cell Dynamics
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2D interaction of non-adherent (immune) cells
2D migration and interaction of non-adherent immune cells
Characterization of the mechanism
Quantitative cell migration assay
Fast and fine analysis of cell migration properties
Macrophage polarization assay (shape)
Modulation of macrophage phenotype by cell shape
Macrophage polarization assay (stiffness)
Modulation of macrophage phenotype with substrate stiffness
IMMUNE CELL INTERACTION ASSAY
2D interaction of non-adherent (immune) cells
4Dcell technology
SmartConfinement Technology
Read-outs
Interaction between cells
Cell type
All non-adherent cells, immune cells, etc.
Standard culture limitation
Non-adherent immune cells poorly migrate on flat surfaces, which limits their interaction with other cells.
In addition, there is an enormous difficulty in qualifying immune cell migration and interaction, since these cells depend on non-adherent confined migration. These events cannot be reproduced in a common petri-dish.
Immune cell interaction assay benefits
It is easier to observe non-adherent (immune) cells in 2D confined spaces than in a 3D matrix. This configuration leads to an increased probability of cellular interaction.
Example
In a confined environment non-adherent immune cells interact at a faster rate [1].
(A) Confinement principle: cells are immobilized between two parallel surfaces with a controlled height. (B) Tracking of cell nuclei position. Results show that cells move more and faster in a confined space enabling to meet neighbouring cells. Scale bars: 100 μm.
IMMUNE SYSTEM IN A WELL
2D migration and interaction of non-adherent immune cells
4Dcell technology
SmartConfinement Technology
Read-outs
Attraction and interaction between cells, quantification of cell migration.
Cell type
Immune cells
Standard culture limitation
Non-adherent immune cells migrate poorly on flat surfaces which can limit the analysis of their behavior.
Immune system in a well assay benefits
Confined between two surfaces (2D), immune cells show restored migration behavior. In these conditions, cells end up meeting more frequently thus enabling their observation and analysis easier than within a 3D matrix.
Example
Migration of mice bone marrow-derived dendritic cells (DCs) in fibronectin coated substrates [3]:
DCs in a confinement assay shows a diffusion coefficient 5 times greater than when plated in common adhesive substrates (substrates coated with extracellular matrix).
FRUSTRATED PHAGOCYTOSIS
Characterization of the frustrated phagocytosis mechanism
4Dcell technology
SmartConfinement Technology
SmartPatternTechnology
Read-outs
Characterization of frustrated phagocytosis, evaluation of the interaction between lysosomes and frustrated phagosomes.
Cell type
Phagocytic cells (e.g. macrophages, neutrophils, dendritic cells, mast cells).
Standard culture limitation
Frustrated phagocytosis cannot be properly observed in a 2D culture due to the 3D process of phagosome formation.
Assay benefits
Using 4Dcell’s assays, molecular mechanisms regulating cellular processes of frustrated phagocytosis can be studied.
On micropatterns, macrophages can target opsonized molecules which are linked to micropatterns, thereby inducing this process [1].
When confined in a small volume, the cell is forced to be spread on the pattern [2].
Example
Frustrated phagosomes formation was observed on RAW cells after opsonization in micropatterns (A), whereas no phagosomes could be detected in the absence of opsonization (B). Human macrophages derived from monocytes (HMDM) were also able to form fp (C) [1].
QUANTITATIVE CELL MIGRATION ASSAY
Fast and fine analysis of cell migration properties
4Dcell technology
SmartConfinement Technology
SmartChannel Technology
Read-outs
Quantification of cell migration speed, persistence and diffusion coefficient
Cell type
All migrating cells (e.g. immune cells)
Standard culture limitation
Non-adherent immune cells poorly migrate on flat surfaces, which can limit the analysis of their behavior. In addition, the 2D random walk of a cell is difficult to analyse.
Traction force assay
When confined, immune cells show restored migration behaviors and can migrate in 1D, 2D or 3D spaces.
This makes their observation and analysis with a microscope easier than within a 3D matrix, enabling the quantification of their exact cell speed, persistence and diffusion coefficient.
Example
MACROPHAGE POLARIZATION ASSAY (SHAPE)
Modulation of macrophage phenotype by cell shape
4Dcell technology
SmartPatternTechnology
Read-outs
Regulation of the functional phenotype
Standard culture limitation
Up to now, it was thought that cytokines and chemokines were the primary regulators of macrophage behavior. However, biophysical cues are also involved in this process
Macrophage polarization assay benefits
Recent studies showed that adhesive cues might modulate proinflammatory versus prohealing activation.
With 4Dcell micropatterns, you can induce an elongation of cells that promote a prohealing phenotype.
Example
Elongation of macrophages by micropatterning.
Cell elongation promotes macrophage polarization toward a prohealing M2 phenotype (increase of arginase-1 level) without influencing inflammatory activation (stable low level of iNOS).
MACROPHAGE POLARIZATION ASSAY (STIFFNESS)
Modulation of macrophage phenotype with substrate stiffness
4Dcell technology
SmartGel Coverslip
Read-outs
Regulation of the functional phenotype
Standard culture limitation
Up to now, it was thought that cytokines and chemokines were the primary regulators of macrophage behavior. However, biophysical cues are also involved in this process.
Cardiac pacemaker cells cytoarchitecture regulation assay
Using 4Dcell gels, macrophages adapt their polarization state, functional roles, and migration mode according to the stiffness of the gel.
Stiff gels prime macrophages towards a pro-inflammatory phenotype with impaired phagocytosis and promote a podosome-dependant slow mesenchymal migration mode, whereas soft gels primes macrophages towards an anti-inflammatory highly phagocytic phenotype and promote a podosome-independent fast amoeboid migration.
Example
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Phenotype depends of the stiffness
Macrophages polarize into pro-inflammatory phenotype represented by proteins (TNFa, MIP1a,IL6) and genes (CCL20, CXCL11) or anti-inflammatory phenotype represented by proteins ( (IL10 ) and genes (CCL22, CCL13, CCL17) based on substrate stiffness.
Sandwich ELISA (bold) and Quantitative RT-PCR (bold italics) for pro- or anti-inflammatory markers.
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Macrophage phagocytosis is increased in soft and medium substrate.
Confocal microscopy images of macrophage phagocytosis of 1 mm latex beads (green) on soft, medium, and stiff gels and quantification.
Nuclei are in blue, actin in purple.
Quantification of the mean velocity of macrophages in mm/hour.