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

1. 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.
2. Prepare agarose casts
   NOTE: 35-microwell casts are used for invasion, immunofluorescence (IF) and cell isolation assays. 81-microwell casts are used for cytotoxicity, IHC, cell isolation and ribonucleic acid (RNA) extraction assays.
   1. After the agarose has been autoclaved, le....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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