Saturated picric acid

After 14 d of culture, hGFs were washed with phosphate buffer saline (PBS) (Biowest, Nuaillé, France), dried overnight at 37 °C in a humidified atmosphere and dried for 24 h at 37 °C in a dry atmosphere. Collagen was stained with 0.1% Sirius red F3BA (Sigma, Saint Louis, Missouri, MO, USA) in saturated picric acid (Sigma, Saint Louis, Missouri, MO, USA) for 1 h. Unbound die was removed by washing with 10 mM HCl (Scharlab, Barcelona, Spain), and dye was solubilized with 100 mM NaOH (Scharlab, Barcelona, Spain). Absorbance was measured with a microplate reader at 540 nm.

For fibrosis quantification, the liver was excised, washed with PBS, and fixed with 10% buffered formaldehyde solution for 24 h. Afterwards, the liver tissue was embedded in paraffin and 6 μm liver sections were obtained. Before staining, paraffin was removed using xylene, xylene/ethanol 1:1, ethanol, ethanol/deionized water 1:1, and deionized water (5 min in each solution). Liver sections were stained in 0.1% Sirius Red F3B (Sigma) with saturated picric acid (Sigma). The relative fibrosis area (expressed as a percentage of total liver area) was analysed in 10 fields of Sirius red-stained liver sections per animal using the morphometry software ImageJ. To evaluate the relative fibrosis area, the measured collagen area was divided by the net field total liver area and then multiplied by 100. From each animal analysed, the percentage of fibrosis area was calculated and the average value presented.

Hamster cheek pouch tissue sections (2 mm thick) were subjected to the picrosirius staining protocol, as previously described in Reference [10 (link)]. Briefly, sections were deparaffinized and rehydrated, immersed in saturated picric acid (Finoric LLC, Houston, TX, USA) (2 g/100 mL) for 10 min. Sections were then stained for 30 min in a 0.1 per cent solution of Sirius red (0.1 g of Sirius red F3BA (Sigma-Aldrich, St. Louis, MO, USA), in 100 mL of saturated picric acid). This was followed by rinsing with tap water, and re-immersion in picric acid for 10 min. Slides were then dehydrated in absolute alcohol (Sigma-Aldrich^®, Saint Louis, MO, USA), clarified in xylene (Sigma-Aldrich^®, Saint Louis, MO, USA), and mounted in synthetic resin (Showa Denko k.k.^®, Tokyo, Japan). Using polarized microscopy (Olympus, New York, NY, USA), this technique resulted in a red or yellow birefringent appearance of type I collagen.

The liver, thymus, and heart were weighed, fixed in 10% formalin overnight, and then embedded in paraffin and sectioned. Five-mm liver sections were stained either with hematoxylin and eosin (H&E) or with 0.1% Sirius red F3B (Sigma, St. Louis, MO, USA) in saturated picric acid (Sigma) [22 (link)]. H&E-stained sections were scored for steatohepatitis grade using the NASH activity score (NAS), which is defined as the sum of the scores for steatosis (0–3), lobular inflammation (0–3), and ballooning (0–2) [23 (link)]. Thus, the results were a continuum ranging from 0 to 8. NAS scores of 0–2 are considered not diagnostic of NASH, while scores of 3–4 are considered possible NASH, and scores of 5–8 are considered diagnostic of NASH. Fibrosis, believed to be a consequence of steatohepatitis, is measured separately from the NAS on a scale ranging from stage 0 to stage 4, with stage 4 indicative of cirrhosis. All liver sections were analyzed and scored in a blinded manner by a trained liver pathologist (Icahn School of Medicine at Mount Sinai Department of Pathology).

After 14 days of cell culture, cells were washed with H₂O, dried for 1 h at RT, followed by incubation for 1 h at −80 °C. Cells were then incubated overnight at 37 °C in a humidified atmosphere, followed by 24 h at 37 °C in a dry atmosphere. Collagen was stained with 0.1% Sirius Red F3BA (Sigma-Aldrich) in saturated picric acid (Sigma-Aldrich) for 1 h at RT. Unbounded dye was removed with 10 mM HCl washes, and dye was solubilized with 0.1 M NaOH. Absorbance was measured at 540 nm. Readings were compared with a calf-skin collagen standard (Calf skin type I Collagen, Sigma-Aldrich) included in the assay. Experiment was run in triplicate (n = 3) for each group and donor.