A three-dimensional engineered heterogeneous tumor model for assessing cellular environment and response.

This protocol describes how to build and implement a three-dimensional (3D) cell culture system, TRACER (tissue roll for analysis of cellular environment and response), that enables analysis of cellular behavior and phenotype in hypoxic gradients. TRACER consists of infiltrating cells encapsulated in a hydrogel extracellular matrix (ECM) within a thin strip of porous cellulose scaffolding that is then rolled around an oxygen-impermeable mandrel for assembly of thick and layered 3D tissue constructs that develop cell-defined oxygen gradients. TRACER differs from other stacked-paper cell culture models because it is assembled from a single-piece scaffold, which facilitates rapid disassembly for analysis of different cell populations and metabolites. The protocol describes how to fabricate TRACER components, cell seeding in the scaffold, and scaffold assembly and disassembly. Furthermore, it provides methods to quantify live, dead, or proliferating cells, as well as gradients of oxygen using the nitroimidazole derivative EF5, in a layer-by-layer analysis with confocal microscopy or by flow cytometry of cells isolated from the TRACER scaffold. Additional methods to isolate live cells from TRACER layers for dose-response analysis with a clonogenic assay, as well as steps to extract RNA or fast-changing metabolites from TRACER layers, are also presented. Finally, we provide alternative steps to establish TRACER co-cultures for assessment of tumor cell invasion and metastasis, in this case in the absence of a hypoxic gradient. Although analysis time varies according to the assay chosen, scaffold fabrication and seeding typically take 2 h, and TRACER assembly takes 20 min on the day following scaffold seeding. The TRACER platform is designed for use by researchers and students who have basic tissue culture experience.