Respiratory

Respiratory diseases have been identified to be an immense health burden worldwide with approximately 1 billion persons suffering from chronic respiratory conditions.

Estimates suggest that:

  • Asthma is the most common chronic disease, affecting about 14% of children globally and rising
  • COPD is the fourth leading cause of death worldwide
  • The most common lethal cancer in the world is lung cancer, which kills more than 1.4 million people each year
  • Respiratory tract infections caused by influenza kill 250,000 - 500,000 people and cost 71-167 billion US dollars annually.
  • - 235 million people suffer from asthma
  • - More than 200 million people have chronic obstructive pulmonary disease (COPD)
  • - 65 million endure moderate-to-severe COPD
  • - 8.7 million people develop tuberculosis (TB) annually
  • - millions live with pulmonary hypertension and
  • - more than 50 million people struggle with occupational lung diseases
  • - each year, 4 million people die prematurely from chronic respiratory disease

The BIG 5 Resporatory Diseases

Available airway cell-based models:

1. Monocultures

  • - Primary Human bronchial epithelial cells (HBEC; healthy and diseased)
  • - Primary Human airway smooth muscle cells (HASM; healthy and diseased)
  • - Primary Human lung fibroblasts (HLF)
  • - Primary Human microvascular endothelial cells - lung (HMVEC-L)
  • - Human Dendritic cells
  • - Immortalised Human Mast Cell Line (LUVA), LAD2, HMC-1
  • - Immortalised bronchial epithelial cells - BEAS-2B
  • - HMC-1 - BEAS2B co-culture: in direct contact
  • - Immortalized human cystic fibrosis bronchial epithelial cell line (CFT1)
  • - Immortalised Human bronchial smooth muscle cell (CFT1)
  • - Peripheral blood mononuclear cells (PBMCs) including monocytes
  • - Peripheral blood granulocytes including eosinophils, neutrophils

2. 3D Airway Model

  • - A highly physiological, three-dimensional cellular system of Human Bronchial Epithelial Cells (HBEpC) differentiated into a pseudostriated epithelium on transwell inserts with a liquid/air interface
  • - 3DAirwayALITM tissue uses primary human airway epithelial cells and fibroblasts grown on an electrospun 3D biological scaffold. These have appropriate epithelial cell barrier function and differentiate into a mucociliated phenotype
  • - Primary HASM cells printed into 3DBioRingTM tissues
  • - 3D HASM (airway smooth muscle) cultures: HASM cells embedded in collagen I
  • - 3D HASM-mast cell co-culture: HASM and mast cells embedded in collagen I
  • - HMC-1 - BEAS 2B 3D co-culture: BEAS 2B were grown to confluence in the top of a Transwell membrane and HMC-1 added to the bottom chamber.
(3DAirwayALITM and 3DBioRingTM are 3D models developed by Aspect Biosystems)

Respiratory Assays:

1. Target validation, qualification

  • - Real time PCR
  • - Gene silencing/knockdown assays
  • - Western Blotting
  • - Immunocytochemistry


2. Proliferation

  • - MTT: indicator of mitochondrial metabolic activity
  • - BrdU: detects 5-bromo-2'-deoxyuridine (BrdU) incorporated into cellular DNA during cell proliferation


3. Cytotoxicity/Viability/Apoptosis

  • - Green Cyanine dye assay: based on the principles of measuring the membrane integrity that occur as a result of cell death
  • - Alamar Blue assay: designed to measure cell proliferation quantitatively by incorporating an oxidation-reduction (REDOX) indicator
  • - LDH Cytotoxicity assay: measures release of a cytosolic enzyme, lactate dehydrogenase, from dead cells
  • - MTT assay: indicator of mitochondrial metabolic activity
  • - Caspase3/7 assay: an assay to detect caspase 3/7 activity as an indicator of apoptosis


4. Migration and Invasion Assays

5. Boyden chamber

  • - Cells seeded at the top chamber and chemoattractant or different cell line in the bottom chamber.
    • - End point: Number of cells migrated to the bottom chamber, compared to controls


6. Collagen gel contraction

3D collagen matrix has also been used in the studies of integrin signaling, cell apoptosis and cytoskeleton reorganization. Since three-dimensional matrix adhesions differ in structure, localization, and function from two-dimensional adhesions; and therefore, three-dimensional cell-matrix interactions may be more relevant biologically

Objective: To assess cell contractivity in vitro and screen cell contraction mediators.

  • Collagen gels impregnated with target cells are resuspended in relevant growth media
  • After polymerization, collagen gels are then pre incubated in the presence or absence of different concentrations of test compound and the contractile stimulant.
  • The collagen gels are then photographed at specified time points over a 3h period by a blinded observer and the gel size as a percentage of the well area is calculated at specific time points using a professional imaging software
  • All gel conditions are performed at least in duplicate.
  • The quantified gel size is used as an indicator of target cell contraction

Example of a representative image of a Collage Gel contraction assay


7. Oxidative stress/DNA damage

  • - 8-oxo-dG immunoassay: biomarker of oxidative DNA damage and oxidative stress
  • - Superoxide Dismutase Assay: a colorimetric assay to detect superoxide Dismutase (SOD) activity in cell and tissue extracts as an indicator of oxidative stress


8. TEER measurement: Trans-endothelial/trans-epithelial electrical resistance (TEER) is the measurement of electrical resistance across a cellular monolayer, and a very sensitive and reliable method to confirm the integrity and permeability of the monolayer.
End-point: Quantitative data - Ohm meter readings.

TEER measurement with chopstick electrodes.The total electrical resistance includes the ohmic resistance of the cell layer RTEER, the cell culture medium RM, the semipermeable membrane insert RI and the electrode medium interface REMI.


9. FITC-Dextran permeability assay: FITC-Dextran molecules leak through cell monolayers where the permeability is compromised.
End-point:
Quantitative data: Determined by measuring the fluorescence of the receiver plate well solution.
Qualitative data: Staining of the monolayer after FITC-Dextran assay.

Representation of FITC-Dextran permeability assay.


10. Vascular Permeability assays (airway endothelial cells)/Membrane Integrity Assays (airway epithelial cells)

  • - Tight junction protein expression (V-Cadherin, occludins and claudins) by immunofluorescence


11. Measurement of Mucin production

  • - Human airway epithelial cells are differentiated using air-liquid interface (ALI) culture method to form mucociliated epithelial cells
  • - Cells are treated in the presence or absence of the test compound and mucus production is then measured by immunoassays


12. Respiratory toxicity and irritation in vitro model

  • - Using differentiated airway epithelial in vitro model characterised by pseudo- stratified epithelium with tight junction formation, numerous apical cilia and apical mucin production
  • - Positive Controls: Bleomycin (Irritation); Triton X-100 (Cell Death)
  • - Biochemical Endpoints: MTT, LDH
  • - Gene and Protein Expression Endpoints: IL-1a, IL-6, IL-8, TNFa, TGFβ
  • - Other Endpoints: Oxidative Stress, Apoptosis


13. Mast cell degranulation assays

  • - HBEC/BEAS 2B and HMC-1 Co-culture model
  • - Sensitisation using either 2.5 µug/ml human IgE or Calcium Ionophore to induce mast cell degranulation
  • - Measure the mast cell tryptase activity using a spectrophotometric method as an indicator of mast cell degranulation
  • - Measure the release of other mediators of allergy and inflammation including histamine, lipoxin A4 by immunoassays
  • - Measure the release of β-hexosaminidase


14. B lymphocytes IgE release assay

  • - Stimulation of B lymphocyte cell line with IL4, IL5 and IL13
  • - Measure IgE release by immunoassays


15. Pathway activation biomarkers

  • - Measure phosphorylation of downstream signalling events
    • Multiplexing immunoassays
    • Western Blot
    • In-Cell ELISA
    • Disease Biomarkers


16. Disease Biomarkers

  • - Measure release of cytokines, chemokines, growth factors and inflammatory mediators (mRNA and protein)
    • Multiplexing immunoassays
    • ELISA