2.7. Measurement of Transepithelial Electrical Resistance (TEER) and Transport of Insulin Using Caco-2 Cell Monolayers

Caco-2 cells were cultured in 175 cm² culture flasks (Thermo Fisher Scientific^TM, Waltham, MA, USA). The culture medium consisted of DMEM supplemented with 10% FBS, 1% MEM-NEAA and 1% antibiotic–antimycotic mixed stock solution. Caco-2 cells were cultured in a humidified atmosphere containing 5% CO₂ at 37 °C. When the cultured Caco-2 cells became sub-confluent, Caco-2 cells were seeded onto Polycarbonate Membrane Transwell^® inserts (pore size: 0.4 µm, growth surface area: 1.12 cm² and membrane diameter: 12 mm, Corning Inc., Corning, NY, USA) at 2.0 × 10⁵ cells/mL. The integrity of Caco-2 cells was monitored by measuring TEER values using a Millicell^® ERS-2 Volt-Ohm Meter (Millipore Corporation, Burlington, MA, USA), and the medium was changed every 2 days. Transepithelial transport studies were performed when the TEER values were more than 500 Ω·cm² (i.e., after 21–23 days). After removing the incubation medium by aspiration, the cells were incubated with modified HBS solution (mHBSS, containing 10 mM HEPES) (pH 6.5) at the apical side and mHBSS (pH 7.4) at the basal side for 60 min at 37 °C. Following preincubation, 0.5 mL of mHBSS (pH 6.5) containing insulin (0.04 mM), with or without Capryol 90 was added to the apical side at the zero-time point, whereas precisely 1.5 mL of mHBSS (pH 7.4) was added to the basal side. At predetermined time points up to 360 min, TEER was measured, followed by sampling 150 μL aliquots from the basal side, and 150 μL of insulin-free mHBSS, which was pre-heated to 37 °C, was added immediately. Each sample was mixed with an equal volume of acetonitrile and was centrifuged (9000× g, 4 °C) for 5 min. The transport of insulin from the apical compartment to the basolateral compartment was measured by HPLC. The apparent permeability coefficients (P_app) for the transported insulin were determined using the following equation:

where P_app is the apparent permeability coefficient (cm/s), Flux is the slope of the linear portion of the cumulative transport profile at the last three points (nmol/min), A is the membrane surface area (1.12 cm²), and C₀ is the initial concentration of the insulin in the donor side (nmol/mL).