3D Bioengineered model of idiopathic pulmonary fibrosis (IPF)

Drug Discovery Assay – reference number: B130

Overview

Testing Information

Assay Description

Alveolar models are constructed using normal lung, or IPF-derived lung fibroblasts. This platform can be used to assess differences in fibroblast behaviour and epithelial-fibroblast crosstalk between the two cellular populations. Pro-fibrotic stimulants can be applied to this model system to trigger key pathological features of pulmonary fibrosis (PF) and idiopathic pulmonary fibrosis (IPF). This platform can also be used to assess the efficacy of anti-fibrotic therapeutic compounds, to alleviate the pathophysiological features of PF and IPF, specifically in relation to fibroblast behaviour and ECM remodelling. 

Test Article Requirements

Test article(s) to be provided by the Sponsor in storable aliquots at required test concentrations with information on the diluent vehicle used. Stock solutions to be prepared in distilled water, PBS or DMSO unless otherwise requested. Sponsor to provide sufficient test article to run the entire study.

Suggested Testing

A minimum of triplicate per condition with a maximum of 60 models per experiment; vehicle controls and positive controls are recommended. Several experiments can be run in a single study to increase model throughput. 

Study Outline

Rationale and Experimental Design

To bioengineer alveolar constructs, our researchers first generate a pulmonary interstitial compartment. Human lung fibroblasts, derived from normal or IPF human lung tissue, are seeded within Alvetex® Scaffold whereby they synthesise and deposit endogenous ECM. Fibroblasts also provide paracrine support to the overlying epithelial tissue in the form of soluble growth factors. A549 cells are then seeded apically upon the interstitial foundation, to form an epithelial compartment, and subsequently generate a microtissue construct of the alveolar epithelium. Once alveolar constructs have matured, soluble test substances are added to culture medium. 

Addition of Test Articles

Schematic representation of culture process. Mature models are transferred to a smaller culture plate, to minimise amount of test article required.

Test articles will be solubilised and added directly to the culture medium for a specified time period, determined by the Sponsor. Typically, compounds have been tested for 24-96 hours; earlier time points are recommended for cytokine release quantification and gene expression analysis, with later time points recommended for assessing ECM remodelling and deposition. Test articles may also be applied to alveolar models in combination with potent pro-fibrotic stimulants, such as TGF-β1 or bleomycin, either therapeutically, prophylactically or simultaneously. Test articles may also be screened alongside known standard-of-care compounds, such as Nintedanib or Pirfenidone.

An example of required conditions for the assessment of two test agents (with pro-fibrotic stimulation):

1.  Vehicle control (if vehicle is not dH₂0, PBS or ≤0.1% DMSO, recommend an additional untouched control)
2. Test Agent 1
3. Test Agent 2
4. Positive control (Pirfenidone or Nintedanib)

Endpoint Analysis

There are a range of endpoints that can be used to assess test article efficacy in this system:

• Fibroblast activation: immunofluorescence staining or PCR analysis for αSMA and FAP activation markers
• ECM deposition and remodelling: immunofluorescent staining for ECM components, quantification of ECM components, quantification of MMP and TIMP secretions, PCR analysis
• Cytokine analysis: secretion quantification, PCR analysis
• Epithelial barrier integrity: histology, TEER measurements, immunofluorescent staining for junctional proteins
• EMT: histology, immunofluorescent staining for epithelial and mesenchymal markers, PCR analysis

Example Data

Figure 1. A pulmonary interstitial compartment is first constructed using normal or IPF-derived human lung fibroblasts, which secrete endogenous ECM proteins. A549 epithelial cells are seeded apically to generate an alveolar epithelial microtissue construct. Scale bars: 50μm.

Figure 2. Normal and IPF bioengineered alveolar models show differences in ECM deposition, ECM remodelling and fibroblast activation.

Figure 3. Pirfenidone attenuates pro-fibrotic effects of TGF-β1 and bleomycin, and reduces total collagen content in IPF models. Scale bars: 50μm. 

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