Insulin from bovine pancreas

Soybean-derived L-alpha-phosphatidylcholine (PC – purity grade ≥97%), A (purity ≥97%), Q (purity ≥95%), K (purity ≥90%), 1,1-diphenyl-2-picrylhydrazyl (DPPH), n-(1-naphthyl)ethylenediamine dihydrochloride, ammonium molybdate tetrahydrate, insulin from bovine pancreas, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich Co. (St Louis, MO, USA). Sodium nitroprusside was purchased from Fluka Chemie AG (Buchs, St Gallen, Switzerland) and sulfanilamide from Friendemann Schmidt (CT Parkwood, Western Australia, Australia). William’s E medium (without phenol red and glutamine) and Glutamax I were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Fetal bovine serum, penicillin−streptomycin, and trypsin were obtained from Nacalai Tesque, Inc. (Kyoto, Japan). Ascorbic acid (AA), hydrocortisone hemisuccinate, and Triton X-100 were obtained from Kollin Chemicals (Malaysia), Santa Cruz Biotechnology Inc. (Dallas, TX, USA), and Alfa Aesar (Heysham, Lancashire, UK), respectively. Dichloromethane (DCM), tetrahydrofuran, dimethylsulfoxide (DMSO), hexane, chloroform, EA, and n-butanol were obtained from EMSURE^® (Darmstadt, Germany). All chemical reagents were of analytical grade unless otherwise stated; extraction solvents were of HPLC/LC–MS grade, purchased from EMD Millipore (Billerica, MA, USA).

Primary hepatocytes were isolated from 6–8-week-old male SD rats (Japan SLC, Inc., Hamamatsu, Japan). Hepatocytes were prepared using a two-step collagenase perfusion method [17] (link), and cell viability was found to be approximately 92% using Trypan blue exclusion test. The culture medium consisted of Dulbecco's Modified Eagle's medium (DMEM), supplemented with 10 mg/L insulin from bovine pancreas (Sigma), 7.5 mg/L hydrocortisone (Sigma), and 60 mg/L l-proline (Sigma); it was hence named as D-HDM [14] (link), [18] . This protocol was reviewed and approved by the Ethics Committee on Animal Experiments of Kyushu University (A29-413-1, 29 Jun 2018).
Before hepatocyte culture, L-ECM pre-gels were adjusted to neutral pH with reconstitution buffer and 10× MEM at a ratio of 8:1:1. Each adjusted sample concentration was equal to four-fifths of their original concentration. Then, freshly isolated hepatocytes were seeded onto L-ECM pre-gels at a seeding density of 2.5 × 10⁵ cells/mL. By incubation under standard conditions (37 °C, 5% CO₂, 95% air) for at least 30 min, hepatocyte-seeded L-ECM gels were obtained. Finally, two hundred microliters of D-HDM was added to each well of a 48-well cell culture plate. The culture medium was refreshed after 1, 3, 5, and 7 days. At least three independent hepatocyte culture experiments were conducted to check the reliability of the results.