DiscussionA central focus for improving drug efficacy in clinical trials over the last decade has been to increase the biological relevance of assays performed early in the drug discovery process. Yet it remains difficult to simulate an in vivo response to drug using an in vitro assay, where the cells are grown on hard plastic or glass substrates, in a two dimensional (2D) format which is not representative of the in vivo cellular environment1 (Pampaloni, 2007). When examining cells within a tissue, it can be observed that cells interact with neighboring cells, and with the extracellular matrix (ECM) to form a communication network. This communication controls a number of cellular processes including proliferation, migration, and apoptosis2 (Bissel, 2002). However, most of the tissue-specific architecture, cell-cell communication, and cues are lost when cells are grown in a more simplified 2D manner. Therefore, more advanced cell culture methods are required to better mimic cellular function within living tissue. 3D cell culture serves to meet this demand by providing a matrix that encourages cells to reorganize into a structure more indicative of an in vivo environment; thereby allowing normal cell-cell and cell extracellular matrix (ECM) interactions to develop in an in vitro environment.
Here we demonstrate an in vitro HTRFï¿½ microplate assay that can quantify total, as well as phosphorylated eIF4E. The assay used a novel 3D culture system called RAFTï¿½ to create cell accumulations termed tumoroids in a cell/collagen hydrogel mix. In addition, the assay was also performed with cells cultured using traditional 2D methods for comparison. Dispensing and removal steps were performed by the MultiFloï¿½ FX Microplate Dispenser, including cell/collagen mix, medium, and reagent dispensing, as well as removal of spent medium and compounds. Detection of the fluorescent signals from the HTRF assay, as well as tumoroid imaging, was performed by the Cytationï¿½3 Cell Imaging Multi-Mode Reader. Validation data generated using RAFT confirms the robustness of the 3D system. Pharmacology comparison data from both methods also demonstrates the validity of using 3D cell culture for cell signaling analyses.