Collagenase c2139

Primary tumor organoids were isolated from murine mammary tumors by stepwise mechanical disruption, enzymatic digestion, and differential centrifugation according to our published protocols (Nguyen-Ngoc et al., 2015 (link)). Briefly, tumors were harvested from MMTV-PyMT or C3(1)-Tag mice with 20-mm tumors, mechanically disrupted with a scalpel, and digested on a shaker for 1 h at 37°C in collagenase solution (DMEM-F12 [10565-018; Gibco Life Technologies] with 2 mg/ml collagenase [C2139; Sigma-Aldrich], 2 mg/ml trypsin [27250-018; Gibco Life Technologies], 5% [vol/vol] FBS [F0926; Sigma-Aldrich], 5 µg/ml insulin [I9278; Sigma-Aldrich], and 50 µg/ml gentamicin [15750; Gibco Life Technologies]). The suspension was centrifuged at 400 g to remove cellular debris and undigested tissue, and the pellet was treated with 2 U/µl DNase (D4263; Sigma-Aldrich). The epithelial organoids were enriched and separated from stromal cells by a series of differential centrifugations, after which organoids of ∼100–500 epithelial cells were obtained. Tumor cell clusters of two to five cells were obtained by taking organoids and further digesting them with 1× TrypLE (12604013; Thermo Fisher Scientific) for 10 min at 37°C. Cells were resuspended in PBS (−Ca²⁺, −Mg²⁺), filtered through a 40-µm filter, and resuspended again at a density of 2 × 10⁶ cells/ml.

The large intestine of each mouse was cut into 1-2 mm pieces. The pieces were stirred twice for 15 min each in PBS containing 3 mmol/L EDTA and twice for 20 min each in RPMI1640 (Hyclone), containing 1 mmol/L EGTA, all at 37 °C, to eliminate epithelium. The remaining pieces were stirred for 90 min at 37 °C in RPMI 1640 (Hyclone) containing 20% fetal bovine serum, 100 U/mL collagenase (C2139; Sigma-Aldrich Corp., St. Louis, MO, United States), and 5 U/mL DNase1 (Sigma-Aldrich Corp). The suspensions were centrifuged, and the pellets were washed. LPL were isolated from the lamina propria (LP)-cell preparations by centrifugation through a 45%-66.6% discontinuous Percoll (Solarbio) gradient at 2500 rpm for 20 min.
LPL (1 × 10⁵/well in 0.2 mL RPMI1640 containing 10% fetal bovine serum, 1% penicillin, and 1% streptomycin) were cultured for 48 h in 96-well plates coated with anti-CD3 (10 μg/mL e-Bioscience, San Diego, CA, United States) and soluble anti-CD28 (1 μg/mL, e-Bioscience) mAb at 37 °C in an atmosphere containing 5% CO₂[25 (link)]. After 48 h, the supernatants were collected and cytokine concentrations assayed by enzyme-linked immunosorbent assay.