Collagenase type 4

To determine the effect of mASCs on DC phenotype and function in vivo, EAE mice were treated i.p. with PBS (control, n = 4) or with allogeneic mASC (n = 4) after the onset of disease (clinical scores: 1-2), and 7 days later, DLNs were isolated and digested with 1.6 mg/mL collagenase type IV and 0.1% DNAse I (Sigma Aldrich) in RPMI1640 medium without supplements at 37°C for 30 minutes. For intracellular TNF-α staining, DLN cells were washed twice with complete RPMI1640 and 2 × 10⁶ cells/mouse were plated in 12-well plates in the presence of 3 μM monensin for 4 h. Cells were gently harvested using cell scrapers, washed using FACS buffer, and processed for intracellular staining as described below. For TNF-α ELISA, CD11c⁺ DCs were immunomagnetically purified using CD11c-microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany) from collagenase type IV-digested DLNs and plated at 2.5 × 10⁵ cells/mL in the presence of LPS (1 μg/mL). Supernatants were collected after 48 hours for cytokine determinations.

TIL were expanded as previously described (14 (link)). Primary bladder tumors or lymph node metastases were minced into ~1–3 mm³ fragments and plated in TIL media consisting of RPMI 1640, 2.05 mM L–glutamine (HyClone, Thermo Fisher Scientific, Waltham, MA), 10% heat-inactivated human AB serum (Omega Scientific, Tarzana, CA), 55 μM 2-mercaptoethanol (Invitrogen), 50 μg/ml gentamicin (Invitrogen), 100 I.U./ml penicillin, 100 μg/ml streptomycin, and 10 mM HEPES Buffer (Mediatech, Manassas, VA) in 24- or 48-well plates with 6000 I.U./ml rhIL-2 (Prometheus). Some cultures were supplemented with 1 ug/ml anti-CD137 agonistic antibody (Urelumab, BMS-663513). All cultures were expanded for 4 weeks and confluent wells were split into additional wells. TIL from each independent fragment was counted. Remaining tumor material was mechanically and enzymatically digested using media containing 2% Collagenase Type IV and a GentleMACS Dissociator (Miltenyi, 130–093-235). Cells were counted by trypan blue exclusion and subjected to subsequent analysis or cryopreserved as functional assay targets. Positive TIL growth was defined as confluency and expansion of the primary well into 2 wells.