Abstract 607: Analyzing cell viability in 3-D tissue models with the ViaLight™ plus cell proliferation and cytotoxicity bioassay
Authors: Buesch Stefanie , Langer John , Schaepermeier Sabine , Hussain Lubna , Bergeron Jeffrey , Vogel Volker , Schroeder Jenny 
Published: July 2016

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. Cells can develop cell-cell and cell-ECM interactions in 3D.
The RAFT™ 3D Culture System uses a collagen matrix at physiologically relevant concentrations. Cells 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. The RAFT™ Absorbers gently remove abundant medium, thus compacting the cell/collagen hydrogel. Additional epithelial or endothelial cells may be added as overlays on top to study co-cultures or more complex cultures.
Another commonly used 3D cell culture model is spheroids. Spheroids are compact aggregates of cells that are generated without the addition of exogenous ECM – e.g. in so-called ultra-low attachment (ULA) plates or by the hanging-drop method.
While 3D cultures more accurately mimic the in vivo cell environment, it might be difficult to analyze cells in 3D due to the dense, tissue-like structure of these cultures compared to 2D cell monolayers. This presentation explains how to measure cell viability in both RAFT™ 3D cell cultures and in spheroid 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 two-step protocol that is used for 2D cell cultures are required for using the assay for 96-well and 24-well RAFT™ 3D cell cultures as well as for spheroid cultures in 96-well ULA plates.
Two different cell types were used in this study: Normal Human Dermal Fibroblasts (NHDFneo) and the colon cancer cell line HCT-116. Both cell types efficiently form spheroids in ULA plates. Also in RAFT™ 3D cultures HCT-116 cells build compact aggregate-like structures, whereas NHDFneo grow in as individual cells interspersed in the collagen scaffold.
By elongating the first step of the Vialight™ Assay, the lysis step, from 10 minutes to 30 minutes, a linear performance of the assay for cell numbers of up to 96,000 cells in the 96-well format and 480,000 cells in the 24-well format could be obtained for RAFT™ cultures. For spheroid cultures an extension of the lysis time to 60 minutes was required to obtain efficient lysis of spheroids with a diameter of up to 400μm, as confirmed by CalceinAM and propidium iodide staining.
In summary this presentation shows that analyzing cells in 3D cultures can easily and routinely be done by slightly adjusting standard 2D cell culture assays like the ViaLight™ Assay.
Citation Format: Stefanie Buesch, John Langer, Sabine Schaepermeier, Lubna Hussain, Jeffrey Bergeron, Volker Vogel, Jenny Schroeder. Analyzing cell viability in 3-D tissue models with the ViaLight™ plus cell proliferation and cytotoxicity bioassay. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 607.