Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON, Canada.
Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, Canada.
Nat Protoc. 2018 Sep;13(9):1917-1957. doi: 10.1038/s41596-018-0022-9.
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.
本方案描述了如何构建和实施三维（3D）细胞培养系统 TRACER（用于分析细胞环境和反应的组织卷），以分析缺氧梯度中的细胞行为和表型。TRACER 由包埋在细胞外基质（ECM）中的浸润细胞组成，该基质位于薄的多孔纤维素支架条内，然后将其卷绕在不透氧的心轴周围，以组装厚且分层的 3D 组织构建体，从而形成细胞定义的氧气梯度。TRACER 与其他堆叠纸细胞培养模型不同，因为它是由单个支架组装而成，这便于快速拆卸以分析不同的细胞群和代谢物。该方案描述了如何制造 TRACER 组件、在支架中接种细胞以及组装和拆卸支架。此外，它提供了使用硝基咪唑衍生物 EF5 在共聚焦显微镜下进行逐层分析或通过从 TRACER 支架中分离的细胞进行流式细胞术来定量活细胞、死细胞或增殖细胞以及氧气梯度的方法。还提供了从 TRACER 层中分离活细胞进行克隆形成测定的剂量反应分析的其他方法，以及从 TRACER 层中提取 RNA 或快速变化的代谢物的步骤。最后，我们提供了替代步骤来建立 TRACER 共培养物，以评估肿瘤细胞侵袭和转移，在这种情况下，不存在缺氧梯度。尽管分析时间根据所选的测定方法而有所不同，但支架制造和接种通常需要 2 小时，而 TRACER 组装在接种支架后的第二天需要 20 分钟。TRACER 平台专为具有基本组织培养经验的研究人员和学生设计。
