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Fibroblast-to-Myofibroblast Transition (FMT) Assay

Assays

  • Fibroblast-to-Myofibroblast Transition (FMT) assay

Models

  • Primary human lung fibroblasts

Timeline

  • 2-4 months

In-vitro assessment of Fibroblast-to-Myofibroblast transition (FMT).

Newcells offers a high-throughput, fast and reliable FMT -assay service to evaluate the efficacy of anti-fibrotic compounds. Our customizable, high-content imaging based assay, is well suited to screening therapeutics targeting fibrosis.

In fibrotic conditions, normal repair processes become defective. Repetitive cellular microinjuries and a heightened immune response leads to chronic activation of resident fibroblasts. When activated, these fibroblasts transition through a pre-activated state to a fully-activated state, a process known as fibroblast-to-myofibroblast transition (FMT). Myofibroblasts show an increased synthesis and secretion of extracellular matrix proteins (ECM), such as collagen I. This excessive ECM deposition within the lung parenchyma leads to progressive scarring, increased lung stiffness and a loss of lung function.

Newcells have developed a high-throughput FMT-assay for the study of lung fibroblast activation and collagen production following stimulation with the well-established fibrotic mediator, TGF-β. Our assay set-up allows for testing of small molecules and biologics, assessing their ability to prevent fibroblast activation and ECM deposition across multiple concentrations. Using duplexed detection and collagen I expression. Our FMT-assay enhances the understanding of compound efficacy to advance drug-discovery programs.

Service outputs

  • Cell number
  • Proliferation capacity (EdU incorporation)
  • Quantification of collagen I expression and deposition
  • Quantification of α-SMA expression
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Human lung fibroblasts stimulated with TGF-β1 for 72hours and immunostained to detect total collagen I (pink) and alpha-smooth muscle actin(α-SMA) (green) as a measure of matrix production and fibroblast activation respectively.

Multiple readouts

Healthy & IPF donors

384-well format

Newcells FMT Assay

Accelerate your lead compound selection by understanding compound efficacy against fibrosis relevant pathology.

1.

Evaluate therapeutic candidates targeting idiopathic pulmonary fibrosis (IPF) and fibrosing lung disease

2.

Determine compound efficacy using high-throughput imaging and reliable quantification

3.

Establish translatability between RNA expression and protein expression changes.

Service overview Close Open

TGF-β1 is well known for its fibrotic effects  in-vivo . Our FMT-assay models the response of fibroblasts to TGF-β1 following a pre-incubation with test articles. Our in-house validation data shows increased cell proliferation, increased incorporation of αSMA into cellular stress fibers and increased expression and deposition of collagen I protein in response to physiologically relevant TGF-β1 concentrations.

Reflective of current literature, we observe an increased cell number in response to physiologically relevant concentrations of TGF-β1 , our assay controls for cell proliferation to ensure changes in expression levels of measured proteins are attributable to test articles. The addition of a macromolecular crowding agent to our media replicates a more  in-vivo like environment and promotes the deposition of secreted collagen I.

Using immunocytochemical detection and high-content imaging our validation data shows a TGF-β1 dose dependent increase in αSMA and collagen I. TGF-β1 effects can be inhibited by the exposure of fibroblasts and ALK5 inhibitors, SB431542 and SB525334.

 

 

 

Effect of TGF-β1 on in-assay fibroblast cell counts Stimulation of human lung fibroblasts with TGF-β1 for 72-hours shows a dose-dependent effect upon total fibroblast number. Graphs showing fibroblast cell count per field of view, based on nuclei staining with Hoechst 33342, for each individual healthy lung fibroblast donor.
TGF-β1 dose-dependently induces αSMA expression Close Open

Human lung fibroblasts cultured in the presence of a macromolecular crowder (MMC) and stimulated with TGF-β1 for 72 hours show a dose-dependent increase in the quantified expression of α-SMA.

Human lung fibroblasts cultured in the presence of a macromolecular crowder (MMC) and stimulated with TGF-β1 for 72 hours show a dose-dependent increase in the quantified expression of α-SMA. (A) Representative images of α-SMA expression, (B) quantified expression levels of α-SMA . Data are combined from three individual human lung fibroblast donors. Asterisk denotes a significant change from the unstimulated control condition.
TGF-β1 dose-dependently induces Collagen I expression Close Open

Human lung fibroblasts cultured in the presence of a macromolecular crowder (MMC) and stimulated with TGF-β1 for 72 hours show a dose-dependent increase in the quantified expression of total and extracellular collagen I.

A) Representative images of total collagen I expression, (B&C) quantified expression levels of collagen I. Data are combined from three individual human lung fibroblast donors. Asterisk denotes a significant change from the unstimulated control condition.
Assay Design
Cell Type

Primary lung fibroblasts

Species

Human

Assay Format

384-well plate with high-content imaging

Assay Readouts
  • Cell number
  • Proliferative capacity (cellular EDU incorporation)
  • Quantification of collagen I expression and deposition
  • Quantification of αSMA expression
Biological Variation

N = 3 validated donors (normal human lung fibroblasts)

Technical Replicates

≥ n = 5 technical replicates/condition

Assay Treatment Window

72 hours

Images

Human lung fibroblasts stimulated with TGF-β1 for 72hours and immunostained to detect extracellular collagen I as a measure of matrix production
Human lung fibroblasts stimulated with TGF-β1 for 72hours and immunostained to detect total collagen I as a measure of matrix production
Human lung fibroblasts stimulated with TGF-β1 for 72hours and immunostained to detect alpha-smooth muscle actin (α-SMA) (green) as a measure fibroblast activation and fibroblast to myofibroblast transition.
Human lung fibroblasts stimulated with TGF-β1 for 72hours and immunostained to detect total collagen I (pink) and alpha-smooth muscle actin(α-SMA) (green) as a measure of matrix production and fibroblast activation respectively.

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