Abstract
(from Protocol to culture and image pancreatic ductal adenocarcinoma tumor slices to study T cell migration, Rodrı´guez-Merced et al, 2023)
Here, we describe a protocol for culture and live cell imaging of tumor slices. This approach studies carcinoma and immune cell dynamics in complex tumor microenvironments (TME) with nonlinear optical imaging platforms. Using a tumor-bearing mouse model of pancreatic ductal adenocarcinoma (PDA), we detail steps to isolate, activate, and label CD8+ T lymphocytes and later introduce them to live murine PDA tumor slice explants. The techniques described in this protocol can improve our understanding of cell migration in complex microenvironments ex vivo.
Method
Prepare a 1.5% agarose solution (Genemate) for support for slicing. Dilute agarose in Tris-acetate-EDTA (TAE) buffer and microwave this solution. When the agarose is completely dissolved, pour solution in a cell culture dish and leave on ice until gel hardens.
Prepare a 6-well and 24-well tissue culture plate with organotypic culture inserts6 with modifications. Begin by filter sterilizing a solution of 100 mM HEPES in 2× PBS. Prepare a 3 mg/mL rat tail collagen type 1 solution by mixing at a ratio of 1:1 of sterile 100 mM Hepes in 2× PBS and complete volume with sterile 1× PBS buffer. Coat 6-well and 24-well inserts with 250 μL and 50 μL, respectively, of 3 mg/mL rat tail collagen solution. Allow collagen mixture to dry in a sterile laminar flow hood for 1–2 h and either use immediately or add 1.1 mL 1× sterile PBS for a 6-well, or 400 μL for a 24-well insert. Store at 4°C for up to two weeks.
Harvest PDA tumor from KPC or KPCT mice, and transfer to a conical tube containing sterile ice-cold PBS with 10 μg/mL STI and 1× P/S for transport.
Using a (feather blade/scalpel), cut a small block from the agarose gel, slightly larger than the tumor, and adhere the agarose to the specimen disk of the vibratome stage using Loctite superglue. Glue the tumor directly in front of the agarose block, which serves as a support for the tumor during slicing. Once the glue dries, fill the tray with ice-cold sterile PBS.
Section the tumor at a 300–350 μm thickness with the vibratome speed set on a 3 mm/s and the vibration frequency set on 8 mm.
Comments