Abstract 307: Determination of cell surface receptors CD44 and ErbB2 in NovaMatrix-3D - a 3D cell culture system

Abstract

Abstract NovaMatrix-3D is an alginate-based cell culture system comprising an alginate foam matrix and an alginate immobilizing solution. Cells are first suspended in a solution of sodium alginate that is then applied to the foam. As the foam absorbs the cell suspension, gelation occurs in situ as calcium ions are donated from the foam which effectively cross-links the added alginate. A hydrogel forms in the pores of the foam entrapping the cells throughout the foam. Cell immobilization occurs at room temperature and physiological pH. Cell localization and multi-cellular structures formed within the transparent gel can be visualized using confocal microscopy after vital staining of cells within the gel. A unique aspect of using an alginate-based foam matrix for 3D cell growth is that cells and intact multi-cellular structures can be retrieved for further analysis by adding calcium chelating agents such as citrate that dissolve the gel. While the retrieval of intact 3D structures is a simple one-step process, it is of interest to evaluate if imaging of cellular receptors can be performed on cells within the 3D hydrogel. Results show that two cell receptors, CD44 and ErbB2, which are upregulated in SKOV-3 cells, can be detected and imaged on multicellular spheroids within the 3D alginate matrix. First, the receptor status of CD44 and ErbB2 were established for SKOV-3 cells growing as a traditional 2D cell culture using flow cytometry. SKOV-3 cells were then immobilized in NovaMatrix-3D using 150 μM RGD-alginate. The presence of RGD has previously been shown to accelerate cell proliferation and 3D cell growth of SKOV-3. After 12 days of growth, multicellular spheroids formed. The alginate hydrogel disks where then treated with FITC-anti-CD44, Pacific Blue-anti-CD44, or an FITC-conjugated Affibody directed against ErbB2. Hydrogel discs were also treated with relevant isotypic controls. Confocal microscopy was used to detect and image cellular receptors. The results show that the CD44 hyaluronan receptor could be detected and imaged using two different antibodies. Furthermore, another cell receptor, ErbB2, was visualized using an Affibody. Therefore, it may not be necessary to retrieve multi-cellular structures from NovaMatrix-3D in order to visualize and image cellular receptors. Traditionally immobilization of cells within an alginate hydrogel has been used to provide immune-protection to cells used in forming an artificial organ, for example pancreatic islets. However, the alginate hydrogel formed in NovaMatrix-3D is comprised of a lower alginate concentration, has a lower, non-saturating concentration of calcium as a cross-linking ion, and has a more open pore structure. These attributes facilitate influx of antibodies which allows in situ immuno-staining and imaging of cellular receptors. The ability to stain cellular receptors on 3D cell structures within an alginate hydrogel make NovaMatrix-3D an even more versatile cell culture system. Citation Format: John Michael Dornish, Therese Andersen. Determination of cell surface receptors CD44 and ErbB2 in NovaMatrix-3D - a 3D cell culture system. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 307. doi:10.1158/1538-7445.AM2015-307