Journal: npj Biomedical Innovations

Article Title: Developing a protocol to counteract spontaneous in vitro activation of intestinal fibroblasts using design of experiments

doi: 10.1038/s44385-026-00079-5

Figure Lengend Snippet: A α-SMA intensity relative to control, B fibronectin secretion, C proliferation, and D viability. Plots show the predicted cell responses generated from predictive model equations for each response variable with all other factors held at their midlevel values. Low, mid, and high levels correspond to 2, 16, and 30 µg/cm² for ECM coatings; 1, 5.5, and 10 µM for vitamin D; and 1, 10.5, and 20 ng/mL for FGF. Solid lines indicate statistically significant effects, whereas dotted lines represent non-significant effects (Table ). Quadratic multiple linear regression models for each response were developed using data from 42 experimental conditions, with six replicates per condition.

Article Snippet: Fibronectin and collagen secretion were quantified using commercially available Human Fibronectin (FN) ELISA Kit (RayBiotech, product # ELH-FN1-1) and Human Pro-Collagen I alpha 1 (COL1A1) DuoSet ELISA (R&D Systems, product # DY6220-05) following the manufacturer’s instructions.

Techniques: Control, Generated

Journal: npj Biomedical Innovations

Article Title: Developing a protocol to counteract spontaneous in vitro activation of intestinal fibroblasts using design of experiments

doi: 10.1038/s44385-026-00079-5

Figure Lengend Snippet: Each graph depicts the effect of paired input variables ( x - and y ‑axes) on A – C α‑SMA fluorescence intensity, D cell proliferation, and E viability ( z ‑axis) with other factors held at mid‑levels. The color scale represents low (dark blue) to high (yellow) predicted response. All panels represent significant interaction effects ( p ≤ 0.05) between the two factors shown on the x ‑ and y ‑axes according to the interaction term p values from the ANOVA table. No significant interaction effects were identified for fibronectin secretion.

Article Snippet: Fibronectin and collagen secretion were quantified using commercially available Human Fibronectin (FN) ELISA Kit (RayBiotech, product # ELH-FN1-1) and Human Pro-Collagen I alpha 1 (COL1A1) DuoSet ELISA (R&D Systems, product # DY6220-05) following the manufacturer’s instructions.

Techniques: Fluorescence

Journal: npj Biomedical Innovations

Article Title: Developing a protocol to counteract spontaneous in vitro activation of intestinal fibroblasts using design of experiments

doi: 10.1038/s44385-026-00079-5

Figure Lengend Snippet: The desirability function algorithm was applied to predict optimal and pessimal culture conditions for minimizing A , B α-SMA intensity and C fibronectin secretion, maximizing D cell viability, and minimizing E proliferation. For each optimization objective, both the optimal (most desirable) and pessimal (least desirable, reverse-ranked) model-predicted conditions (Table ) were tested experimentally. Opt.α-SMA, Opt.FN, Opt.Prolif, and Opt.Viab refer to the conditions predicted when each response was individually optimized. Opt.all refers to the simultaneous optimization of all responses, and Opt.all (-prolif) refers to the simultaneous optimization of all responses excluding proliferation. Untreated cells cultured on TCPS served as the control. Data are presented as mean ± SD ( n = 5). Scale bar indicates 100 μm. Two-way ANOVA followed by Tukey’s multiple comparisons test. Different letters above bars indicate statistically significant differences between groups ( p < 0.05); matching letters denote no significant difference ( p > 0.05).

Article Snippet: Fibronectin and collagen secretion were quantified using commercially available Human Fibronectin (FN) ELISA Kit (RayBiotech, product # ELH-FN1-1) and Human Pro-Collagen I alpha 1 (COL1A1) DuoSet ELISA (R&D Systems, product # DY6220-05) following the manufacturer’s instructions.

Techniques: Cell Culture, Control

Journal: npj Biomedical Innovations

Article Title: Developing a protocol to counteract spontaneous in vitro activation of intestinal fibroblasts using design of experiments

doi: 10.1038/s44385-026-00079-5

Figure Lengend Snippet: A Immunofluorescence staining of the cell cytoplasm using Cell Mask (green) and Phalloidin (red). B , C Quantification of cell area and aspect ratio. Gene expression analysis of D ACTA2 , E MYLK , and F MYH11 via qRT-PCR. G Immunofluorescence staining of α-SMA (red) and FAP (green), with cell nuclei counterstained with DAPI (blue). Quantification of the staining intensity of the myofibroblastic protein markers H α-SMA, I FAP. Gene expression analysis of J COL3A1 and K COL1A1 via qRT-PCR. L Quantification of secreted collagen I using ELISA. Data are presented as mean ± SD ( n = 3–5 replicates). Scale bar indicates 100 μm. One-way ANOVA followed by Tukey’s multiple comparisons test. * p < 0.05, ** p < 0.01,*** p < 0.001 and **** p < 0.0001 compared to the control and ## p < 0.01, ### p < 0.001, and #### p < 0.0001 compared to TGF-β 1 .

Article Snippet: Fibronectin and collagen secretion were quantified using commercially available Human Fibronectin (FN) ELISA Kit (RayBiotech, product # ELH-FN1-1) and Human Pro-Collagen I alpha 1 (COL1A1) DuoSet ELISA (R&D Systems, product # DY6220-05) following the manufacturer’s instructions.

Techniques: Immunofluorescence, Staining, Gene Expression, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Control

Journal: npj Biomedical Innovations

Article Title: Developing a protocol to counteract spontaneous in vitro activation of intestinal fibroblasts using design of experiments

doi: 10.1038/s44385-026-00079-5

Figure Lengend Snippet: A Quantification of cell viability. Gene expression analysis of B ACTA2 , C MYLK , and D MYH11 via qRT-PCR. E Immunofluorescence staining of α-SMA (red) and FAP (green), with cell nuclei counterstained with DAPI (blue). Quantification of the staining intensity of the myofibroblastic protein markers F α-SMA and G FAP. Gene expression analysis of H COL3A1 and I COL1A1 via qRT-PCR. Quantification of ECM protein secretion for J collagen I and K fibronectin using ELISA. Data are presented as mean ± SD ( n = 3–5 replicates). Scale bar indicates 100 μm. One-way ANOVA followed by Tukey’s multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 compared to the control and # p < 0.05, ## p < 0.01, #### p < 0.0001 compared to TGF β 1 -treated group, for comparisons shown.

Article Snippet: Fibronectin and collagen secretion were quantified using commercially available Human Fibronectin (FN) ELISA Kit (RayBiotech, product # ELH-FN1-1) and Human Pro-Collagen I alpha 1 (COL1A1) DuoSet ELISA (R&D Systems, product # DY6220-05) following the manufacturer’s instructions.

Techniques: Gene Expression, Quantitative RT-PCR, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Control