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Immune System Models

Tunable In Vitro Assays for Immune Cell Dynamics

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Immune cell interaction

2D interaction of non-adherent (immune) cells

Immune system in a well

2D migration and interaction of non-adherent immune cells

Frustrated phagocytosis

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].

Confined-immune-cells-1024x518.png

(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).

Confinement-application.jpg

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-phagocytosis-on-micropatterns-1.jpg

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

Microchannels-application.jpg

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).

macrophages-by-micropatterning.jpg

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

  • 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.

fig-1-pro-vs-anti-inflammatory-status.png

Sandwich ELISA (bold) and Quantitative RT-PCR (bold italics) for pro- or anti-inflammatory markers.

  • Macrophage phagocytosis is increased in soft and medium substrate.

macrophage-phagocytosis-gels-graph.png

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.

macrophage-migration-gels.png

Quantification of the mean velocity of macrophages in mm/hour.

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