Respiratory Disease Modeling and the Use of Primary Cells

Oxygen is the key to life and something most take for granted every day. For those whose every breath is painful or a struggle, getting oxygen into their blood stream is anything but simple. Respiratory disease is a common and significant cause of illness and death globally and includes:

• Chronic respiratory diseases such as asthma and COPD, which are characterized by high inflammatory cell recruitment. These conditions are generally incurable, but various treatments are available to patients to help dilate their airways, reduce inflammation, reduce overproduction of mucus and ultimately relieve their shortness of breath.
• Restrictive lung disease such as idiopathic pulmonary fibrosis (IPF), which is characterized by a loss of lung compliance due to a build-up of scar tissue (fibrosis). About 5 million people are affected globally, with an average life expectancy following diagnosis of approximately four years. There are currently two drugs available which both act to slow down the development of fibrosis.
• Genetic disorders such as cystic fibrosis (CF), which is an autosomal recessive multi-organ disease. It is characterized primarily by defective electrolyte transport in airway cells and excess mucus secretion with poor clearance leading to chronic infection and inflammation. There is currently no cure for CF; treatment focusses on controlling and minimizing the recurrent respiratory infections the patients suffer from.
• Malignant lung tumors, which are responsible for 15% of all cancers diagnoses and 30% of all cancer deaths. Non-small cell lung cancers account for 85% of lung cancers and are currently largely insensitive to chemotherapy.

Understanding the mechanisms and key biology behind these diseases is critical to future development of new therapies and possible cures. Access to diseased primary cells from patients diagnosed with respiratory disease (from asthma and IPF to NSCLC) provides a convenient and highly relevant biological tool to assess genetic and phenotypic changes from normal healthy donor tissue.

In healthy individuals, the airway epithelium functions as a physical barrier for potential pathogens and an immune-modulator capable of releasing cytokines and chemokines to fight infection. The airway is formed of pseudostratified columnar epithelium containing the following:

• Goblet cells (GC) which secrete mucus to maintain epithelial moisture and trap pathogens or air particulates;
• Basal cells (BC) which have the capacity to differentiate into other cells types to restore a healthy epithelial cell layer;
• Ciliated cells (CC) which move to carry mucus and trapped particulates up and out of the airways.
• A) Diagram of an ALI culture, (B) Transverse paraffin section of an ALI culture stained with H&E, (C) Scanning electron micrograph of an ALI culture showing well-differentiated ciliated cellsA well-established 3D model for looking at airway epithelial cells in vitro is the air liquid interface (ALI) model, since increased exposure to air is crucial for the development of a well-differentiated epithelium and associated barrier. Isolated primary cells from healthy or diseased donors are seeded onto a basement membrane and grown to confluency as submerged cultures before generation of the ALI. To achieve ALI, the cells are exposed to specialized media basolaterally and apically to humidified air containing 5% CO₂. Cells grown under these conditions will differentiate into pseudostratified columnar epithelium over 21-28 days and contain a mixed population of cells including goblet, basal and ciliated cells. This model can be used to study:
  • Airway remodeling – compounds can be added or removed from the specialized media or applied to the apical surface to alter the differentiation process. For example, particulates found in cigarette smoke can lead to the mucus-secreting ciliated cells being replaced by stratified squamous epithelium.
  • Mucus secretion – as the differentiated cells are capable of mucus production, this can be manipulated by compounds and measured downstream. Overproduction of mucus is common in many chronic respiratory diseases and also in cystic fibrosis, where production of viscous mucus results in airway plugging which can be responsible for fatalities associated with this disease.
  • Inflammatory responses – as the cells are capable of enlisting an immunological response this can be manipulated by the addition of drugs in isolation or combination and the resultant levels of downstream cytokine and chemokine release measured.

  In addition to the functional endpoints described above that can be investigated using 3D models of the lung epithelium, the ALI approach also allows for the whole basement membrane with the differentiated cells in-situ to be paraffin wax embedded and sectioned for immunohistochemistry analyses. Alternatively, the differentiated cells can be lysed for downstream RNA or DNA analysis to interrogate disease-relevant gene expression levels.

  ALI cultures, whilst more time consuming to establish than some other 3D lung models, such as spheroids, precision-cut slices and explant cultures, have certain advantages over these other models. ALI cultures differentiate into the functional epithelial cell types described, can be maintained long term in culture, culture conditions can be modified to recapitulate specific environments, whilst retaining the potential to create co-cultures with other cell types such as fibroblasts or inflammatory cells.

  Advantages and disadvantages of this 3D organotypic ALI model ^{adapted from [1]}:

  Advantages	Disadvantages
  Long term cultivation (several months)	Typically derived from a single cell type – lack of complexity and cellular interaction
  Opportunity for direct application e.g. aerosols, particulates
  Perfusion possible
  Applicable for a variety of functional studies
  Can be sourced commercially High data reproducibility Low batch-to-batch variability Well-characterized Several established pathologies available Longer-term cultivation (up to 12 months)	Established commercial models not so easily modified

  The development of physiologically improved 3D models is an area of great interest among researchers to enable better drug response prediction. BioreclamationIVT provides products and services to support respiratory disease research including the following:

  • Access to diseased tissues and biofluids from a range of respiratory diseases
  • Custom cell isolations
  • 2D primary cell models
  • 3D primary cell models – OrganDOT™ platform, ALI cultures
  • Primary cell inventory from asthma, COPD, CF, IPF, PF and lung cancer
  • Target localization and validation – single and multiplexed IHC and ISH
  • Gene expression services (qRT-PCR, mutation analysis)
    
    In summary, physiologically relevant models of differentiated airway epithelium can be created using human primary cells from a range of different respiratory diseases to provide a suitable in vitro approach to investigating the mechanism of action of a variety of therapeutic interventions for disease treatment.
    
    

     

     

     

    [1] K Zscheppang et al., Human Pulmonary 3D Models for Translational Research. Biotechnol. J. 2018, 13, 1700341

    February 12, 2018 Categories: Tags: