Glutamin

Huh-7.5 cells (kind gift from Charles Rice), HEK-293T kidney cells (ATCC CRL-3216), TE-671 cells (ATCC CRL8805), A549 cells (kind gift from P. Boulanger), VeroE6 cells (ATCC CRL-1586), EBL cells (kind gift from Fabienne Archer), MDBK cells (European Collection of Authenticated Cell Cultures (ECACC)) were grown Dulbecco’s modified minimal essential medium (DMEM, Invitrogen, France) supplemented with 100 U/mL of penicillin, 100 µg/mL of streptomycin and 10%. of fetal bovine serum.
PHH (BD Biosciences) were centrifuged in F12-HAM medium (Sigma Aldrich) and seeded overnight in collagen-coated plates in BD Gentest seeding medium supplemented with 5% FCS. 16 h later, PHH were washed and cultured with a culture medium for PHH (DMEM F12, Sigma Aldrich) supplemented with 10% FCS, 1 µg/mL BSA, 5 µg/mL bovine insulin, 1 × 10⁻⁶ M Dexamethasone (Sigma Aldrich), 1 × 10⁻⁸ M 3.3 trilodo-L-thyronin, 5 µg/mL apotransferrin, 1% of non-essential amino acids (Gibco), 1% of Glutamin (Gibco) and 1% Penicillin-Streptomycin solution (Gibco).

Induced pluripotent stem cells (iPSCs) were differentiated into hepatocytes according to the previously published protocol^{35 (link)}. In brief, cells were seeded into growth factor-reduced Matrigel-coated 6-well plates (2 million cells per well) in STEMdiff definitive endoderm basal medium (StemCell Technologies) containing Supplements A and B and cultivated for one day. From day 2 to 4, medium was exchanged daily and only contained Supplement B. At day 5, cells were detached and re-seeded into 96-well plates with a density of 12,000 cells per well. Medium was changed to Differentiation medium for hepatic specification: High-glucose DMEM/F12 (Gibco); 10% KOSR (Gibco); 1% Glutamin (Gibco); 1% Non-essential amino acids (Gibco); 1% Pen/Strep (Gibco); 100 ng/ml Human Growth Factor (peprotech); 1% DMSO; 10 µM Rock inhibitor Y27632 (Millipore).
From day 7 to 13, medium was changed to Differentiation medium without Rock inhibitor and refreshed daily. At day 14, medium was changed to Cultivation medium, which contained 10⁻⁷ M Dexamethasone (Sigma Aldrich) instead of Human Growth Factor and DMSO. This medium was also changed daily and used during treatments and further analysis.
To assess the level of iPSC differentiation into hepatocytes, expression levels of markers were analyzed on mRNA level by qRT-PCR: ALB, ABCC2, CYP1A1, HNF4A and RXRA.

Thirty islets per well were transferred to an extracellular-matrix-coated 96-well plate (Biological Industries) in complete RPMI1640 and incubated overnight in a 5% CO₂ humidified atmosphere at 37°C. Medium was removed and NMRI islets were washed in KRBH (115 mM NaCl; 4.7 mM KCl; 2.6 mM CaCl₂; 1.2 mM KH₂PO₄; 1.2 mM MgSO₄; 5 mM NaHCO₃ (all from Merck); 1% penicillin/streptomycin (vol/vol) (Gibco); 20 mM Hepes (Gibco); 0.02 mM Glutamin (Gibco); 0.2% (wt/vol) BSA (Sigma), pH 7.4). Islets were then incubated in KRBH added 3 mM D-glucose for 0.5+1 h and KRBH added 15 mM D-glucose with/without LPS or 150 pg/ml IL-1β and 5 ng/ml IFNγ for 1 h. Buffer was collected from the last 3 mM and 15 mM incubations, filtered through a 96-well filter plate (Multiscreen-DV, Millipore) and stored at −20°C until analysis. Each experiment was normalized to the insulin release at 15 mM ( = 100%). Thus, when calculating the average from the 3 independent experiments, the variation of the 15 mM stimulation will be 0.15 mM glucose corresponds to the lowest concentration of glucose giving maximum insulin release.

HUVECs were isolated from human umbilical cords. The umbilical cords were collected and kept at 4 °C in PBS for a maximum of 3 days. To harvest the ECs, the vein was cannulated and flushed with sterile PBS. Thereafter, pre-heated (37 °C) trypsin-EDTA (Gibco) was incubated for 15 minutes in 37 °C PBS after which the vein was flushed with PBS again. The resulting cell suspension was spun down (1500 rpm, RT, 10 minutes), resuspended in Endothelial growth medium-2 (Lonza), and plated on 1% gelatin coated T75 flasks, cultured at 37 °C, 5% CO₂. Experiments were performed with cell passage 1–3.
MVEC were a kind gift from Prof. M.J.T.H. Goumans, Leiden University Medical Center, Leiden, the Netherlands. MVECs are human pulmonary microvascular endothelial cells and were culture in microvascular endothelial cell medium-2 (EGM-2-MV, Lonza).
Human embryonic kidney cells (HEK293T) were cultured in DMEM (Gibco) supplemented with 100 U/ml penicillin, 100 U/ml streptomycin, 300 μg/ml glutamin (all from Gibco) and 10% fetal calf serum (Cambrex).

Dendritic cells were differentiated from CD14 positive peripheral blood mononuclear cells by washing in PBS (Gibco, Carlsbad, CA, USA) and ficoll density gradient centrifugation at 350 g for 10 min twice and adding 5 ml RPMI medium supplemented with 1% Pen/Strep and 1% FCS (Biochtrom, Berlin, Germany). Cells in the cell suspension are subsequently counted using 20 µl Tryptan Blue (Sigma-Aldrich, St. Louis, MO, USA) in a hemocytometer. Seeding of 2.0 × 10⁶ cells/ml and incubating at 37 °C under 5% CO₂ was performed for 2 h. The supernatant and non-attached cells were discarded. 500 µl basal Iscove’s medium was added to the cells supplemented with 1% Pen/Strep, 1% glutamin, 5% HSA, (all Gibco, Carlsbad, CA, USA) 100 ng/mL IL-4, 100 ng/mL GM-CSF (both Peprotech, Rocky Hill, NJ, USA) with medium change every other day for 6 days at 37 °C before TPE-FLIM imaging. The purity was regularly controlled and reached approx. 90%.