Primary human preadipocytes are widely used to study adipogenic differentiation. However, adipose tissue remodeling extends beyond changes in lipid accumulation: under pro-fibrotic conditions, preadipocytes can also acquire myofibroblast-like features.
We therefore developed a preadipocyte fibrosis assay that combines two imaging-based phenotypic readouts in primary human cells: fibrogenic activation and adipogenic differentiation. This approach enables both responses to be evaluated across donor-derived cultures within the same experimental platform.
Assay setup
The assay was established using four primary human preadipocyte cultures representing two adipose tissue depots and donors with and without diabetes:
| Donor | Depot | Donor status |
| EPI-02 | Epicardial | Non-diabetic |
| EPI-05 | Epicardial | Diabetic |
| PRE-02 | Visceral | Non-diabetic |
| PRE-05 | Visceral | Diabetic |
For the adipogenic arm, cells were differentiated using insulin, dexamethasone, IBMX, L-thyroxine, and ciglitazone. Lipid droplets, nuclei, and cell morphology were visualized by fluorescence imaging, and lipid-positive area was normalized to cell number.
For the fibrogenic arm, cells were exposed to TGF-β as pro-fibrotic stimuli. Fibrogenic activation was assessed by α-smooth muscle actin (α-SMA) immunofluorescence.
Establishing a robust fibrogenic readout
Secreted COL1A1 was initially evaluated as a readout of fibrogenic activation. However, the ELISA showed insufficient reproducibility across donor cultures because measured collagen levels were influenced by differences in cell number and donor-dependent proliferation.
We therefore moved to an imaging-based α-SMA readout, quantifying the percentage of α-SMA-positive cells rather than total secreted protein. This provided a more reproducible quantitative endpoint for comparing fibrogenic responses across donor cultures.
The α-SMA assay achieved a Z′ factor >0.7, indicating robust separation between assay controls and supporting its use for quantitative phenotypic analysis.
The assay reveals donor-specific responses
Seven days after pro-fibrotic stimulation, the assay resolved different α-SMA response patterns across the four donor-derived cultures.
| Donor | α-SMA response | Phenotype |
| PRE-02 | Minimal induction | Resistant |
| EPI-02 | Minimal induction | Resistant |
| PRE-05 | Measurable increase | Slightly inducible |
| EPI-05 | Strongest response; elevated baseline | Strongly inducible, high baseline |
The assay captured distinct donor-specific fibrogenic responses, ranging from resistant to strongly inducible phenotypes. EPI-05 showed the strongest response to pro-fibrotic stimulation, together with elevated baseline α-SMA expression.
Adding adipogenic response as a second phenotype
Lipid accumulation was quantified in parallel as lipid-positive area normalized to cell number. Adipogenic capacity differed among donor-derived cultures, but TGF-β treatment did not consistently reduce lipid accumulation across all four models: a donor can show a strong fibrogenic response without a corresponding change in adipogenic differentiation. Measuring both endpoints within the same assay therefore captures phenotypic responses that neither readout would reveal alone.
What this assay enables
The established workflow provides a quantitative primary-cell platform for characterizing donor-dependent preadipocyte phenotypes and evaluating responses to pro-fibrotic stimulation.
- Donor characterization and selection for downstream studies
- Quantitative assessment of TGF-β-induced fibrogenic activation
- Parallel measurement of fibrogenic and adipogenic phenotypes
- Compound testing in primary human preadipocytes
- Phenotypic studies in fibrosis and cardiometabolic research