Development of a novel multicellular 3-dimensional blood brain - - PowerPoint PPT Presentation

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Development of a novel multicellular 3-dimensional blood brain - - PowerPoint PPT Presentation

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Development of a novel multicellular 3-dimensional blood brain barrier in vitro model Vetsuisse Faculty, Institute of Veterinary Physiology and Zurich Center for Integrative human physiology. University of Zurich, Zurich, Switzerland Omolara

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Development of a novel multicellular 3-dimensional blood brain barrier in vitro model

Vetsuisse Faculty, Institute of Veterinary Physiology and Zurich Center for Integrative human physiology. University of Zurich, Zurich, Switzerland Omolara Ogunshola, PhD

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The blood brain barrier (BBB) protects the brain environment

Consists mainly of 3 cells;

  • Endothelial cells
  • Astrocytes
  • Pericytes

From Abbott 1989

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  • The complexity of the brain and location of the BBB has enabled us only little

understanding of induction and formation of the BBB

  • Further signals that disrupt or modulate BBB function are poorly understood
  • Thus good simplified model systems of BBB are sorely needed.

In vitro modeling

  • 2D models are most common but are limited
  • 3D models less common and have various advantages

REDUCE, REFINE and REPLACE (3Rs)

  • Animal testing is difficult, invasive and requires many experiments
  • Interpretation of the results is often difficult
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Anatomy of cellular junctions in brain endothelial Anatomy of cellular junctions in brain endothelial cells cells

PECAM-1 Cadherin βcat Actin Occludin PECAM-1 Cadherin ZO1

Adherens junctions Tight junctions

Flk-1 VEGF Tie2 Ang-1

Occludin Endothelial cell Luminal (blood) face Abluminal (brain tissue) face

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Hypoxia-induced loss of barrier function is visualized by Hypoxia-induced loss of barrier function is visualized by delocalization of proteins involved in cell-cell contact delocalization of proteins involved in cell-cell contact

PECAM-1 staining 1% O2 0.1% O2

10 m

β-catenin staining 1% O2 0.1% O2 0h 24h 48h

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Modified Boyden chamber is used to measure transendothelial electrical resistance (TEER) in presence of astrocytes and pericytes.

Technical approach Technical approach

Contact model Non-contact model

Cell culture Cell culture

Rat brain endothelial cells (RBE4; Roux et al. 1994, J. Cell physiol.) Rat primary astrocytes (perinatal animals) Rat primary pericytes (12-16 week old rats)

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Hypoxia disrupts RBE4 tight junctions resulting in decreased TEER values

Short term Long term

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Endothelial tight junction disruption is differentially modulated by astrocytes and pericytes.

Short term Long term

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  • Endothelial cells are resistant to long term hypoxia
  • Astrocytes provide significant protection during hypoxic stress
  • Pericytes have a biphasic response to hypoxic exposure
  • Presence of all 3 cell types is only advantageous during long

term stress.

Conclusion

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Why do we need a new 3D BBB model? Why do we need a new 3D BBB model?

Answer: 2D models are limited

  • 2D model does not accurately reflect in vivo situation
  • Interactions between cells are different.
  • BBB consists of 3 cells not just 2 (pericytes overlooked)
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Novel 3-dimensional BBB model? Novel 3-dimensional BBB model?

Advantages

  • Allow cell-specific interactions to occur that can be directly observed
  • Observe different stages of barrier formation - induction and maturation
  • Understand BBB function disruption (after hypoxic/oxidative stress)

Disadvantages

  • No blood flow!
  • Rest of brain millieu absent
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Technical approach Technical approach

Acid soluble collagen solution

NaOH

Basic insoluble collagen solution

cell suspension Spot droplets Overlay with media

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RBE4 form tube-like structures RBE4 form tube-like structures in vitro in vitro

2 DIV 4 DIV 6 DIV

Phase contrast Fluores- cence Endothelial cells (PECAM-1) in green

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Tie-2 (Angiopoeitin receptor) PECAM-1 (adhesion molecule)

RBE4 form tube-like structures RBE4 form tube-like structures in vitro in vitro

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Imaging of RBE4 tube-like structures Imaging of RBE4 tube-like structures after sectioning show hollow tubes after sectioning show hollow tubes

Endothelial cells (PECAM-1) in red

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Astrocytes Astrocytes interact with RBE4 tube-like interact with RBE4 tube-like structures in 3D culture structures in 3D culture

Endothelial cells (PECAM-1) in red, astrocytes (GFAP) in green

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Astrocytes Astrocytes interact with RBE4 tube-like interact with RBE4 tube-like structures in 3D culture structures in 3D culture

From Abbott 1989 Confocal image

Endothelial cells (PECAM-1) in red, astrocytes (GFAP) in green

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How can we follow tube formation and cellular interactions in real time?

Direct fluorescence with lipophilic fluorescent dye

+

DiI/PKH

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Live imaging of RBE4/astrocyte interactions using lipophilic dyes

Endothelial cells in red, astrocytes in green

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Double-staining in fixed collagen droplet Live imaging in collagen droplet (lipophilic dye)

Resolution after lipophilic-labeling is reduced compared to fixed imaging

  • Need to use confocal and 2-photon microscopy to get better images and to observe

cells more closely.

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What about pericytes??

Pericytes survive in collagen and label with lipophilic dyes. Some interactions take place.

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Summary

Developing a novel 3D model of BBB which can be observed in real-time.

  • Observe cell interactions
  • Stage-specific development
  • Can be subjected to hypoxic/oxidative stress to observe barrier breakdown
  • Other cells can easily be added to the matrix
  • Eventual in drug testing (alterations in cell interactions and movement)?

Genetic profile analyses to identify cell-specific genes and signalling pathways that are modulated during the different stages of model stabilisation.

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Acknowledgements

Abraham Al-Ahmad

  • Prof. Max Gassmann (University of Zurich)
  • Prof. Joseph Madri (Yale University)
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Tube formation depends Tube formation depends

  • n collagen density
  • n collagen density

2.5 mg/mL collagen 3.25 mg/mL collagen

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Astros/endos ratio is important for interactions

Indirect fluorescence (endos in red and astros in green) 3:1 ratio 4:1 ratio

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RBE4 cells - Normoxia - Actin

(Z-stack 10 plan - 18µm thickness)

Z 3/10 Z 6/10 Z 9/10