Ex Vivo T Cell Stimulation in the Pancreatic Tumor: A Method to Increase Cytokine Production in T Cells

Abstract

Source: Spear, S., et al. Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity. J. Vis. Exp.(2019).

This article describes the technique for ex vivo T cell stimulation to increase the intracellular concentration of cytokines such as interferon-gamma or IFN-gamma and tumor necrosis factor-alpha or TNF-alpha in the T cell. These cytokines  mediate an effective anti-tumor response and can have a therapeutic role in the tumor. 

Protocol

1. Digestion of pancreatic tumors

1. Place the dissected pancreatic tumor, metastatic site tumors, or healthy pancreatic tissue in ice-cold PBS, and store on ice.
   1. Use forceps to transfer the tumor onto a Petri dish.
   2. Add 5.0 mL of digestion medium (2 mg/mL Collagenase, 0.025 mg/mL DNase RPMI) into a 50 mL tube; store on ice to prevent enzyme activity commencing.
      NOTE: This protocol uses Collagenase Type V, which has an activity of ≥1 units/mg FALGPA and > 125 collagen digestion units (CDU)/mg solid. Collagenase and DNase aliquots can be stored at -20 °C and thawed on ice before use. When both are c....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
70 μm pore-size cell strainer	Fisher Scientific	11597522
anti-CD107a PE (clone 1D4B)	Biolegend	121612	1:100 to culture media
Bovine Serum Albumin (BSA)	Sigma-Aldrich	A4503
Cell Stimulation Cocktail (500x) (phorbol 12-myristate 13-acetate (PMA) and ionomycin)	eBioscience	00-4970-03	Final concentration PMA 0.081 μM, ionomycin 1.34 μM
THP1 Human Monocytic Leukemia Cells	ATCC	TIB-202
Collagenase Type V from Clostridium histolyticum	Sigma-Aldrich	C9263	2 mg/mL in media
Dimethyl sulphoxide (DMSO)	Sigma-Aldrich	D2650-100mL
DNase (Deoxyribonuclease I from bovine pancreas Type II-S) stock 10 mg/mL in 0.15 M NaCl	Sigma-Aldrich	D4513	Final concentration in digestion media 0.025 mg/mL
Foetal calf-serum (FCS)	GE Healthcare	A15-104	10% in RPMI
Hamilton syringe 700 series, 25 μL volume, 22s gauge needle bevel tip	Fisher Scientific	10100332
Penicillin/streptomycin	PAA	15140122	100 units/mL Penicillin, 100 μg/mL Streptomycin
Protein transport inhibitor cocktail (500x) (brefeldin A and monesin)	eBioscience	00-4980-03	1x Final concentration Brefeldin A 10.6 μM, monensin 2 μM
RPMI-1640 (containing 0.3 g/L Glutamine)	Sigma-Aldrich	R8758
Surgical Scalpel Blade No.10	Swann-Morton	501
Trypsin-EDTA Solution 10x	Sigma-Aldrich	594-18C	Trypsin (0.1%) EDTA (0.4%) final concentration
U-bottomed 96 microwell plate	VWR	734-2080