Insulin transferrin selenium ethanolamine its x

Immortalized human podocytes were cultured in RMPI 1640 (Corning, Tewksbury, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Corning, Tewksbury, MA, USA), 1% 100 X Penicillin Streptomycin L-Glutamine (PSG) (Corning, Tewksbury, MA, USA), and 1% Insulin-Transferrin-Selenium-Ethanolamine (ITS-X) (Gibco, Gaithersburg, MD, USA) [39 (link)]. Proliferating podocytes were cultured in a humidified atmosphere with 5% CO₂ at 33 °C and the media was changed twice per week and cells were passaged at ~70–80% confluence. Differentiation was induced by placing the cells at 37 °C under the same atmospheric conditions, for 14 days. Differentiated cells were treated with puromycin aminonucleoside (PAN) (Sigma-Aldrich, St. Louis, MO, USA) at 25 ug/mL or with pioglitazone (Alfa Aesar, Tewksbury, MA, USA) at 10 μM. Control cells received treatment of vehicle dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA). Cells were harvested after 24 h and total RNA isolated.

Mouse kidney fibroblasts were isolated and cultured as previously described with some modifications^{46 (link)}. Briefly, the mouse kidneys were harvested 3 days after UUO surgery, and the cortex diced and digested in 0.05% trypsin/EDTA for 10 min at 37 °C. The kidney pieces were placed in gelatin-coated dishes and incubated in DMEM/F12 with Glutamax (Gibco; Thermo Fisher Scientific) supplemented with 20% FBS, 1× Insulin-Transferrin-Selenium-Ethanolamine (ITS-X; Gibco), 5 µM Y-27632 (Enzo Life Sciences, Farmingdale, NY), and 1% penicillin/streptomycin for 3 days, after which the kidney pieces were gently removed and the medium was changed. The culture medium was renewed twice weekly. To examine the effect of TGF-β stimulation on gene expression, fibroblasts were cultured in 6-well plates to confluency, followed by serum starvation for 12 h before adding 10 ng/mL human recombinant TGF-β (R&D systems, Minneapolis, MN). Similarly, we stimulated the rat kidney fibroblasts cell line (NRK-49F; American Type Culture Collection, Manassas, VA) with TGF-β or BMP7 (R&D systems) but serum starvation was performed for 24 h before adding stimulation. Cells were harvested after 24 h and total RNA was extracted using TRIzol reagent.

Lyophilized PNIPAAm‐g‐CS and alginate MPs were sterilized by soaking in 70% isopropanol and exposure to UV light. Then, 5% wt/vol PNIPAAm‐g‐CS was prepared in NP differentiation medium containing high glucose DMEM (Gibco), 1% FBS (Gibco), insulin‐transferrin‐selenium‐ethanolamine (ITS‐X) (Gibco), 100 μM l‐ascorbic acid‐2‐phosphate (Sigma‐Aldrich), 1.25 mg/mL bovine serum albumin (BSA) (Sigma‐Aldrich), 0.1 μM dexamethasone (Sigma‐Aldrich), 40 μg/mL l‐proline (Sigma‐Aldrich), 5.4 μg/mL linoleic acid (Sigma‐Aldrich), antibiotic‐antimycotic (Gibco) and 100 ng/mL of GDF6 (PeproTech).⁶² (link) Pelleted ADMSCs were combined with the solutions prepared in media of 5% PNIPAAM‐g‐CS + alginate MPs (S‐50) or 5% PNIPAAm‐g‐CS (P‐0). The final cell density in each of the formulations was 5 × 10⁶ cell/mL. Last, ~100 μL of each cell‐seeded formulation was dispensed into a 48 well plate and gelled before adding 500 μL of NP differentiation media on top. Media was refreshed every other day and the formulations were cultured for 14 days.