Human ab serum hs

Human AB serum (HS) was purchased from Sigma- Aldrich (Brøndby, Denmark); RPMI-1640 with GlutaMAX and AIM-V medium were obtained from Invitrogen (Nærum, Denmark). RhIL2 (Proleukin) was from Novartis (Basel, Switzerland), and OKT3 (anti-CD3) antibody was from Cilag AG (Schaffausen, Switzerland). Pulmozyme was purchased from Roche (Basel, Switzerland). Allogeneic peripheral blood mononuclear cells (PBMCs) (or feeder cells) were obtained from buffy coats from healthy donors. Solu-Cortef (hydrocortisone sodium succinate) was obtained from the local hospital pharmacy. Fetal bovine serum (FBS) was from Gibco (Nærum, Denmark). Complete medium (CM) consisted of RPMI-1640 with GlutaMAX, 25 mM HEPES pH 7.2, 100 U/ml penicillin, 100 μg/ml streptomycin and Fungizone^® (Bristol-Myers Squibb, New York, NY, USA) 1,25 lg/ml supplemented with 10% HS and 6000 IU/ml of rhIL2. Rapid expansion medium (RM) consisted of AIM-V medium and Fungizone^® 1,25 lg/ml supplemented with 6000 IU/ml rhIL2.

Clinical efficacy was assessed by Flour-18-deoxyglucose-positron emission tomography/CT (FDG-PET) scans at baseline, before hospitalization, at week 6 and 12 after TIL infusion and every third month thereafter until disease progression. Objective responses were evaluated according to RECIST V.1.1.
Blood samples for immune monitoring were collected at baseline, before hospitalization, at discharge and at following evaluation visits. Blood was collected in heparinized tubes and was kept for a maximum of 4 hours until handling according to standard operating procedure. In brief, peripheral blood mononuclear cells (PBMCs) were separated using centrifugation on a Lymphoprep (Takeda, Roskilde, Denmark) density gradient. Vials of PBMCs were cryopreserved in medium containing 90% heat inactivated human AB serum (HS; Sigma Aldrich) and 10% dimethyl sulfoxide using controlled-rate freezing (Cool-Cell, Biocision) in a −80°C freezer and the next day moved to a −140°C freezer until further processing.
Serum samples were collected before TIL infusion, 2 hours after TIL infusion and every second day until discharge. Within maximum 4 hours, serum tubes were spun at 3000g for 10 min. Serum was aliquoted and immediately transferred to a −80°C freezer and subsequently stored at −140°C until further processing.

Sliced decellularized tissues were equilibrated with DMEM 1% P/S (no serum) at 4 °C overnight. Decellularized tissues were removed from DMEM 1% P/S and excess liquid removed. Decellularized tissue slices were placed into the wells of 96-well plate. To the decellularized tissues, 25 μL of isolated monocytes were added to the center of the tissue at seeding density of 2 × 10⁵/25 μL. The monocytes were incubated with the decellularized tissue at 37 °C for 2 h to allow the cells to attach to the tissue. After 2 h, 200 μL DMEM supplemented with 10% human AB serum (HS) (Sigma-Aldrich) and 1% P/S was added carefully to the cultures, not to disturb the tissue adhered cells. Cultures were maintained for 14 days.