A Three-Dimensional Co-culture Model of Infiltrating T Cells in Tumor Spheroids

Abstract

Source: Lin, Y., et al. Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture. J. Vis. Exp. (2020).

This video demonstrates a method to establish a three-dimensional tumor spheroid and T cell co-culture. An agarose cast with microwells containing tumor spheroids is prepared, followed by the addition of T cells. After incubation, this setup allows studying the interaction of T cells with tumor spheroids in a controlled three-dimensional environment.

Protocol

1. Generation of spheroids Prepare and autoclave 2% agarose in 1x phosphate-buffered saline (PBS) (e.g., 1 g agarose in 50 mL of 1x PBS) and autoclave 35- and 81-microwell rubber molds. CAUTION: Avoid using low-melting agarose for generating the agarose casts for immunohistochemistry (IHC) processing. Prepare agarose casts NOTE: 35-microwell casts are used for invasion, immunofluorescence (IF) and cell isolation assays. 81-microwell ca…

Materials

3D Petri Dishes	Microtissues Inc	Z764019 & Z764051	referred to as "rubber molds" in the protocols; 81-microwell & 35-microwell molds
8-well Chamber Slides	Lab-Tek	154534
Agarose Type I, low EEO	Sigma-Aldrich	A6013
DMEM, fetal bovine serum	ThermoFisher	11965092, 16000044	referred to as "cell culture medium" in the protocols
RPMI	ThermoFisher	11875093	for T-cell culture medium