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Validation of a Novel Tumoroid - Based Cell Culture Model to Perform 3D in vitro Cell Signaling Analyses

Comparison of Results Generated with an in vitro Cell Signaling Assay using Cells Cultured in 2D and a Novel 3D Cell Culture System

Brad Larson and Peter Banks, BioTek Instruments, Inc., Winooski, VT
Grant Cameron, TAP Biosystems, Royston, Hertfordshire, UK
Nicolas Pierre, CisBio US, Inc., Bedford, MA

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Over the last decade, a central focus for improving drug efficacy in clinical trials has been to increase the biological relevance of assays performed early in the drug discovery process. Yet it remains difficult to simulate a drug’s in vivo response using an in vitro assay. In vitro assays typically use cells grown on two-dimensional (2D) hard plastic or glass substrates, which are not representative of the true in vivo cell environment. In tissue, cells interact with neighboring cells and with the extracellular matrix (ECM) to form a communication network that affects many cellular processes including proliferation, migration and apoptosis. In a simplified, in vitro 2D environment, most of the tissue-specific architecture, cell-cell communication and cues are lost. Therefore the need exists for advanced culture methods that better mimic cellular function within living tissue.

Three-dimensional (3D) cell culture methods, in comparison, provide a matrix that encourages cells to organize into structures more indicative of the in vivo environment, thereby developing normal cell-cell and cell-ECM interactions in an in vitro environment. Here, we demonstrate an in vitro HTRF® microplate assay from Cisbio Bioassays (Bedford, MA, USA) that assesses total and phosphorylated Eukaryotic translation initiation factor 4E (eIF4E) using a novel 3D cell culture system. The RAFT™ (Real Architecture For 3D Tissue) cell culture system from TAP Biosystems (Royston, Hertfordshire, UK) creates a 3D hydrogel environment using the most abundant matrix protein, collagen type I, at physiologically relevant concentrations. Within this culture certain cells can assemble into higher order structures, termed tumoroids, driving cell-cell and cell-matrix interactions. The HTRF® assay was also performed with cells cultured using traditional 2D methods for comparison.

All liquid dispensing and removal steps were performed by the MultiFlo™ FX Microplate Dispenser from Bio Tek Instruments (Winooski, VT, USA), including dispensing of cell/collagen mix, medium, and reagents, and removal of spent medium and compounds. BioTek’s Cytation™ 3 Cell Imaging Multi-Mode Reader detected the HTRF® assay’s fluorescent signals, and also imaged the tumoroids. Validation data generated using RAFT confirms the robustness of the 3D cell culture system and its ease of use with automated platforms. Pharmacology comparison data between the 2D and 3D methods also demonstrates the validity of using 3D cell culture for cell signaling analyses.

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