Gelatin from bovine skin

Gelatin from bovine skin (Gel; type B, Bloom ~75), low molecular weight chitosan (CS; 92.2% degree of deacetylation), and polyvinyl alcohol (PVA; 89 kDa, 99.8% hydrolysis) were purchased from Sigma Aldrich (St. Louis, MO, USA). For multilineage differentiation: transforming growth factor-β (TGF-β; Peprotech, London, UK. Cat. 100-21), bone morphogenetic protein 4 (BMP-4; Peprotech, Cat. 120-05), dexamethasone, trypsin and ascorbic acid (Gibco, Carlsbad, CA, USA). For enzymatic digestion: type II collagenase (Worthington Biochemical, Lakewood, NJ, USA). For viability assays: live/dead viability/cytotoxicity kit for mammalian cells (Invitrogen L3224, Carlsbad, CA, USA).

C57BL/6 mice (H‐2^b, CD45.2⁺) were purchased from Shimizu Experimental Animal Laboratory (Shizuoka, Japan), and congenic CD45.1⁺ C57BL/6 mice were purchased from RIKEN BioResource Center (Ibaraki, Japan). All animals were maintained under specific pathogen‐free conditions. Mouse iPSCs were provided by RIKEN BioResource Center.²¹ Cells were cultured in advanced Dulbecco's modified Eagle medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 50% supernatant from mouse foetal fibroblasts in Dulbecco's modified Eagle medium (Sigma‐Aldrich, St. Louis, MO, USA) with 10% fetal bovine serum. Cells were cultured in the presence of 10³ U/ml leukemia inhibitory factor (ORF Genetics Ltd., Kópavogur, Iceland) and 0.1 mM 2‐mercaptoethanol (2ME) (Sigma‐Aldrich) in 0.1% gelatin from bovine skin (Sigma‐Aldrich)‐coated 10‐cm dishes. They were passaged every 2 or 3 days by treatment with 0.25%Trypsin‐EDTA (Thermo Fisher Scientific) for 1 min at 37°C. Aliquots of cells in the proliferative phase were harvested and maintained in liquid nitrogen until use in experiments. The Animal Experimentation Committee of Kansai Medical University reviewed and approved the animal studies. EL‐4 (H‐2^b, CD45.2⁺), a leukemic T‐cell line derived from C57BL/6 mice,²², ²³ was used for the in vivo study.

According to a report by Alarcón-Segovia et al.36 (link), 2.5 g gelatin from bovine skin (Sigma-Aldrich, USA) with glycerol and MilliQ water in a 12-well culture plate into a thickness of 1-2 mm were maturated 15 minutes as forming polymer. 5 μl of 100 μg/ml coffee leaf (Coffea Arabica) extract, 75% (weight/weight [w/w]) Ethanol, or H₂O (control) were applied separately on skin-like gelatin membranes at room temperature from 15 minutes to 1 hour and added into 2 μl pseudo-viral particles (MOI=0.2) for 2 hours. Subsequently, they were added with 50 μl of medium for the Vpp infection assay.

Gelatin from bovine skin, phytic acid sodium salt hydrate, and Pluronic^® F-127 were purchased from commercial sources Sigma-Aldrich (Sigma Aldrich, St. Louis, MO, USA; and Sigma-Aldrich Chemie GmbH, Steinheim, Germany, Merck, Darmstadt, Germany) without additional purification processes. The heating of mixtures was performed in a Mettler-Toledo Easymax 102 Advanced Synthesis Workstation using 25 mL closed reactor tubes.

xanthine oxidase from bovine milk, luminol sodium salt, xanthine, oxypurinol, PBS tabs, Na-EDTA salt, gelatin from bovine skin, penicillin/streptomycin, trypsin-EDTA, Trolox, and 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium perborate, boric acid, NaOH, and FeCl₂ were from Carlo Erba (Milan, Italy). M200 medium, low serum growth supplements, and fetal bovine serum, RNaseOUT, were purchased from Thermo Fisher Scientific (Waltham, MA, USA). RNeasy Mini Kit was from QIAGEN (Hilden, Germany). Primers for RT-PCR were purchased from IDT (Coralville, IA, USA). Cell counting kit-8 (CCK8) and LDH assay kit were purchased from Dojindo Molecular Technologies (Rockville, MD, USA). SuperScript® III First-Strand Synthesis SuperMix and EXPRESS SYBR® GreenER™ qPCR SuperMix were purchased from Life Technologies (Carlsbad, CA, USA). All the other chemicals and solvents were of the highest analytical grade.

Primary MEFs were prepared as described previously (Spector, 1997 ; Besson et al., 2004b (link)) from p27+/+, p27CK−/CK− or p27−/− embryos. MEFs were immortalized by infection with retroviruses encoding the human papilloma virus E6 protein and hygromycin selection. HeLa (RRID:CVCL_0030), HEK 293 (RRID:CVCL_0045), A-375 (RRID:CVCL_0132) and A549 (RRID:CVCL_0023) cells were obtained from Cell Lines Services. These cells were authenticated by short tandem repeat profiling. All cells were routinely tested to be free of mycoplasma contamination by DAPI staining.
All cells were grown at 37°C and 5% CO₂ in DMEM (Sigma), 4.5 g/l glucose supplemented with 10% fetal calf serum, 0.1 mM nonessential amino acids and 2 µg /ml penicillin-streptomycin. When indicated, tissue culture vessels were coated with 0.2% gelatin from bovine skin (Sigma G1393) for 1 hr. MEFs were infected with different plasmids using retroviruses produced in Phoenix ecotropic cells transfected by the calcium phosphate method. HEK293 cells were also transfected by the calcium phosphate method for 24 hr. HeLa cells were transfected using JetPrime (Polyplus transfection) according to manufacturer’s instructions. For siRNA transfections in MEF E6 cells, we used Interferin (Polyplus transfection) according to manufacturer’s instructions.