Dispase 1

Mouse aorta ECs were isolated and cultured^{14 (link)}. In brief, thoracic aorta was isolated from 3-week-old Tie2-Cre;Pdgfrb^fl/fl and Pdgfrb^fl/fl mice. Aortic rings were embedded in Matrigel-coated dishes and cultured for 5 days. After rinsing with phosphate-buffered saline (PBS), the rings were removed, and remaining cells incubated with 2 U/ml Dispase I (Gibco, 17105-041) for 20 min at 37 °C. After centrifugation at 500 × g for 10 min, the cell pellets were washed with PBS, and cells cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 medium supplemented with 25 mg/ml EC growth supplement (Sigma) and 5% fetal bovine serum (FBS) at 37 °C in a humidified air atmosphere with 5% CO₂.

Mouse aortic ECs were isolated from Cdh5-Cre^ERT2;Il6^fl/fl mice as previously described^{35 (link)}. In brief, thoracic aorta was isolated from 3-week-old mice and cut into pieces. Aortic rings were embedded in phosphate-buffered saline (PBS)-prewashed Matrigel and then incubated in DMEM/F-12 medium containing 5% FBS for 5 days. After rinsing with PBS, the rings were removed and the remaining cells were incubated with 2 U/ml Dispase I (Gibco, 17105-041) for 20 min at 37 °C. After centrifugation at 500 × g for 10 min, the cell pellets were washed with PBS. ECs were also isolated from mouse brain. Single-cell suspension was prepared and subjected to MACS with anti-CD31 antibody-conjugated magnetic beads (Miltenyi Biotech, 130-091-935). Cells were cultured in DMEM/F-12 medium supplemented with 25 μg/ml EC growth supplement (Sigma) and 5% FBS. All cells were used between passages 2 and 4.

Lamina propria-resident immune cells from small intestine were isolated by digesting intestinal tissue with Collagenase VIII (Sigma-Aldrich). Shortly, Payer’s patches were removed, and epithelial layer was dissociated by incubating tissue pieces twice in 2mM EDTA/PBS for 20 min at 37 °C with shaking (180 rpm). Afterwards, single cell suspension was prepared by digesting small intestinal tissue in complete RPMI medium (10% FCS, 1% P/S, 1% Glu) containing Collagenase VIII (0.6 mg/ml) for approximately 15 min at 37 °C with shaking (180 rpm). Lamina propria-resident immune cells from large intestine were isolated by digesting intestinal tissue with Collagenase D (Roche), Collagenase V (Sigma-Aldrich), Dispase I (Gibco), and DNase I (Roche). Here, epithelial cells were removed by incubating tissue pieces twiese in 2mM EDTA/PBS for 15 min at 37 °C with shaking (180 rpm). Next, single cell suspension was prepared by digesting large intestinal tissue in complete RPMI medium (10% FCS, 1% P/S, 1% Glu) containing Collagenase D (1.25 mg/ml), Collagenase V (0.85 mg/ml), Dispase I (1 mg/ml), and DNase (30 U/ml) for a maximum of 45 min at 37 °C with shaking (180 rpm). Cell number of single cell suspensions (2% FCS/PBS) were assessed, and cells were kept at 4 °C for immediate flow cytometry analysis.

A total of nine PDAC cultures were used in this study (Table 1). PDAC organoid cultures (n = 6) were established as previously described [15 (link)]. Briefly, tumor specimens were minced and digested with Collagenase II (5 mg/mL, Gibco) and Dispase I (1.25 mg/mL, Gibco) in human complete medium [15 (link)] at 37°C for a maximum of 2 h. The resulting material was further digested with TrypLE (Gibco) for 10 min at 37°C, embedded in growth factor reduced Matrigel (Corning) and cultured in human complete medium (described in [15 (link)]). Tissue digestions from three additional PDAC specimens were directly seeded on tissue-culture vessels for initiation of monolayer cell cultures using the following medium: Advanced DMEM/F12 medium supplemented with HEPES (1X, Gibco), Glutamax (1X, Gibco), Primocin (1 mg/mL, Invivogen), mouse Epidermal Growth Factor (50 ng/mL, Gibco), Dexamethasone (3 nM, Sigma), and 5% Fetal Bovine Serum (Gibco). Both monolayer cell cultures and organoids were routinely tested for the presence of mycoplasma using MycoAlert Detection Kit from Lonza in accordance with the manufacturer’s instructions.

Five C57 mice were sacrificed in accordance with NIH guidelines, and the corneas were isolated under a surgical microscope. The isolated corneas were incubated with 2% dispase I (Gibco, Grand Island, NY, USA) for 1 hour at 37 °C. The epithelial layers and basement membrane were removed with a scalpel under a surgical microscope. Then the remaining corneal tissue was chopped and incubated with 2% collagenase in Dulbecco’s Modified Eagle Medium (DMEM) for 2 hours at 37 °C. Dissociated cells were harvested by centrifugation at 1,500 rpm for 10 minutes, seeded on 35-mm² dishes in DMEM containing 10% fetal bovine serum (FBS), and cultured until 90% confluence. Isolated primary keratocytes were used up to passage 6.