T75 cm2 flask

To determine the optimal cell seeding density for BMSC isolation, nucleated cells from samples 01 to 04 were resuspended at 0.08 × 10⁵/cm², 0.4 × 10⁵/cm², and 2.0 × 10⁵/cm² in 10 mL of α-MEM supplemented with 20% FBS and plated in triplicate in T-75 cm² flasks (Corning Incorporated). After plating, the cells were allowed to adhere for 3 days in a humidified atmosphere of 5% CO₂ at 37°C. Then, the nonadherent cells were removed, the adherent cells were washed three times with PBS, and the medium was changed. Thereafter, the adherent cells were allowed to proliferate for 11 additional days. Complete medium exchange was performed every 3 days. At day 14, a 5 mL aliquot of the culture medium was collected for bacterial contamination testing. The cells were washed twice with PBS and harvested with recombinant trypsin (TrypLE® Express, Invitrogen, Carlsbad, CA, USA). The cell number was determined by manual counting with a Neubauer chamber. The cell viability was assessed by the Trypan Blue exclusion method. If the cell viability was <70%, the cells were discarded and expansion was stopped. For samples 05 to 14, nucleated cells were plated only at 0.4 × 10⁵/cm², and the isolation was performed as described.

The rat insulinoma β cell line INS1 (clone 832/13) was a kind gift from Dr Chris Newgard.³³ INS1 β cells were cultured in T‐75 cm² flasks (Corning) in regular RPMI‐1640 medium (Gibco, Invitrogen) unless indicated otherwise, with 2 mmol/L L‐glutamine (Gibco, Invitrogen), 10% heat‐inactivated foetal bovine serum (Atlanta, Optima), 100 U/mL penicillin, 100 μg/mL streptomycin, 10 mmol/L HEPES, and 50 mmol/L β‐mercaptoethanol (all from Gibco, Invitrogen) at 37°C, 5% CO₂. Cells were passaged once a week when reaching 90% confluence and used between passages 30 and 65. In certain experiments, cells were serum‐starved with minimum medium constituted by RPMI‐1640 medium (Gibco, Invitrogen), 2 mmol/L L‐glutamine (Gibco, Invitrogen), 1 mmol/L Sodium Pyruvate (Gibco, Invitrogen), 0.1% bovine serum albumin (BSA; fat‐free, Sigma), 100 U/mL penicillin, 100 μg/mL streptomycin, 10 mmol/L HEPES (Gibco, Invitrogen) and 3 mmol/L glucose. β‐mercaptoethanol was omitted in the medium in all conditions and assays of stress and death. A dose of 0‐10 nmol/L human C‐peptide (Phoenix Pharmaceuticals) was selected based on previous published reports.¹⁰, ¹¹, ¹², ¹³, ¹⁵, ¹⁷

Caco-2/15 cells were cultured at 37 °C and 5% of CO₂ in EMEM medium (Wisent Inc., Canada) containing 10% fetal bovine serum (FBS; Wisent Inc.), 1% penicillin/streptomycin and 1% non-essential amino acids (GIBCO, USA) as described previously^{64 (link),65 (link)}. Cells were maintained in T-75 cm² flasks (Corning Inc., USA) until they reached 80–90% of confluence and were trypsinized. For individual experiments, enterocytes were seeded at the density of 1 × 10⁶ cells/well on polycarbonate 24.5-mm transwell filters with pores of a diameter of 0.4 µm (Corning Inc., USA). Differentiation occurred when enterocytes were cultured for 15 days post-100% confluence.