Abstract 334: Creation and analysis of advanced in vitro cancer models in the real architecture for 3D tissue (RAFT™) culture system
Authors: Schaepermeier Sabine , D'Souza Theresa , Menezes Aurita , Schwartz Claudia , Schroeder Jenny 
Published: September 2020

Abstract
Conventional in vitro assays are based on cells grown on two-dimensional (2D) substrates, which are not representative of the true in vivo cell environment. In tissue environments, cells interact with neighboring cells and the extracellular matrix (ECM). Three-dimensional (3D) cell culture methods allow cells to grow in structures more resembling the in vivo environment. The RAFT™ 3D Culture System uses a collagen matrix at physiologically relevant concentrations. In the presented applications, cells from the colon cancer line HCT 116 and neutralized collagen are mixed and subsequently incubated at 37°C to allow the formation of a cell-seeded hydrogel. Specialized RAFT™ Absorbers are placed on top of the hydrogels. Those absorbers gently remove abundant medium, thus compacting the cell/collagen hydrogel prior futher culture. Additional cell types like fibroblasts, endothelial or epithelial cells may be added to allow the creation of more complex models. While 3D cultures more closely mimic the in vivo cell environment, it is often difficult to apply transient transfection methods to these dense, tissue-like structures. Here we give a guideline how to use the electroporation-based Nucleofector™ Technology for creating transiently transfected cells for further culture in the RAFT™ System. HCT 116 was harvested and transfected with pmaxGFP™ Vector in suspension using the 4D Nucleofector™ System X-Unit. Subsequently, cells were embedded in RAFT™ Cultures. Overall transfection efficiencies and cell viabilities were comparable between RAFT™ 3D Cultures and standard 2D cultures. Transfection efficiencies of more than 90% were observed in both formats. GFP expression was still detectable 4 days after transfection. Innovative gene-editing tools like CRISPR-Cas nuclease-based systems could be used to obtain more long-term transgene expression. Another challenge is the analysis of cells in dense, tissue-like structures compared to 2D monolayer culture. HCT 116 cells formed compact aggregate-like structures in RAFT™ 3D Cultures that resembled small tumors in vivo. Therefore, we developed a protocol for immunocytochemistry that allows effective antibody staining of HCT 116 containing RAFT™ Cultures. In addition, we will explain how to measure cell viability in RAFT™ Cultures with the ViaLight™ Plus Cell Proliferation and Cytotoxicity BioAssay. The ViaLight™ Assay is based on the bioluminescent detection of cellular ATP as a measure of cell viability. Only minor modifications of the standard 2D cell culture protocol are required for using the assay for RAFT™ Cultures. By elongating the lysis step of the Vialight™ Assay from 10 minutes to 30 minutes, a similar performance of the assay was observed for HCT 116 containing 2D and RAFT™ Cultures. In summary, we show how easily genetically modified in vitro 3D cancer models can be generated in the RAFT™ 3D Cell Culture System and how such models can be analyzed using standard methods like immunocytochemistry and the ViaLight™ Viability Assay.
Citation Format: Sabine Schaepermeier, Theresa D'Souza, Aurita Menezes, Claudia Schwartz, Jenny Schroeder. Creation and analysis of advanced in vitro cancer models in the real architecture for 3D tissue (RAFT™) culture system [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 334.