Trypsin Inhibitors

Having established the cell lines as described, cells were routinely expanded for experimental work on laminin coated T75 cm² or T175 cm² tissue culture flasks (Nunc Easyflask) in B27 medium [DMEM:F12 containing B27 neural cell supplement mix (Gibco), L-Glutamine (2 mM, Gibco), heparin (10 Units/ml, Sigma) and Gentamicin (50 μg/ml, Gibco)] in the presence of bFGF (10 ng/ml, Invitrogen) and EGF (20 ng/ml, Sigma) – referred to as growth medium. All cells in culture were maintained at 37°C in a humidified atmosphere of 95% air/5% CO₂. It is important not to allow the cells to become confluent (> 80% confluency) at any time. Cells were passaged every three to four days using trypsin and trypsin inhibitor solutions. Briefly, cells were rinsed with HBSS without Ca²⁺/Mg²⁺ and then incubated in trypsin solution for 5–15 min until the cells detached. Twice the volume of trypsin inhibitor was added and the cells centrifuged at 500 g × 5 min. The cell pellet was resuspended in fresh medium and plated in a freshly laminin coated flask(s) at a density of ~10000 cells/cm². For consistency and practical reasons, all experiments presented in this study were carried out on cells between passages 15 and 30. However, karyotype analyses were carried out on longer-term cultures and found to be normal.

Directly after sacrifice, pancreata of one-month old mice were injected with 2 ml Collagenase P solution (1.33 mg/ml Collagenase P (Roche) in HBSS (Gibco)), cut out, minced with a scalpel and gently shaken for 30 min at 37 °C in 5 ml Collagenase P solution. All subsequent steps were performed at 4 °C in a laminar flow cabinet and all centrifugation steps were carried out for 3 min at 180 x g. Cells were resuspended in 10 ml 5% FBS in HBSS and incubated 10 min for sedimentation of the cellular fraction. Supernatant was aspirated carefully, and the pellet was washed 3 times with 5% FBS in HBSS. Cells in 10 ml 5% FBS in HBSS were transferred into a new tube through a 100 µm cell strainer, slowly laid over 20 ml 30% FBS in HBSS and centrifuged. Cells were resuspended in 2 ml recovery medium (acinar cell medium, see below, with 30% FCS), incubated at 37 °C for 1 h, centrifuged and resuspended in a 1:1 mixture of acinar cell medium (containing 0.1% bovine serum albumin, 0.2 mg/ml soybean trypsin inhibitor (Sigma), 1% ITS premix (Corning), 50 µg/ml bovine pituitary extract (ThermoFisher), 0.1% FBS, 0.5% penicillin/streptomycin, 0.25 µg/ml Fungizone antimycotic (ThermoFisher) in Waymouth’s medium (Gibco) and rat tail collagen type I (Corning). Per pancreas, cells were seeded into 16 wells of a 48-well plate on a previously prepared collagen layer (final collagen concentration 2.5 mg/ml) and covered with another collagen layer before adding acinar cell medium. Medium was changed every 24 h.
Five days after seeding, images were acquired with AxioVision Rel 4.8 software and the percentage of ductal structures of the total amount of acinar explants was determined by counting 5 microscopic fields of view at 100x magnification for each pancreas. Quantification was blinded to the genotype.