Background

In vitro differentiation assays are a remarkable tool that enables us to analyze the potency of cells without relying on in vivo models. Cells in these assays can be manipulated for gene expression to, for instance, analyze their differentiation ability. While previous protocols have described the differentiation potential of primary salivary gland cells in vitro, our protocol is optimized for the use of the SIMS cell line (Laoide et al., 1996; Maimets et al., 2016). SIMS cells express cytokeratin 7 and 19, which is characteristic for a ductal cell type. Although adult acinar and myoepithelial cells were found in vivo to preserve their own cell population through self-duplication (Aure et al., 2015; Song et al. 2018), in some cases duct cells can differentiate into acinar cells in vivo, such as after radiation injury (Lombaert et al., 2008; Weng et al., 2018). Thus, utilization of SIMS cells allows us to target and analyze the self-renewal and differentiation effects of ductal cells under specific in vitro controlled conditions. Our recent work shows that SIMS cells have the ability to differentiate into unique populations of acinar, myoepithelial, and duct cells in 3D differentiation conditions, that normally produce, modify and secrete saliva in vivo (Lombaert et al., 2011; Athwal et al., 2019). This protocol describes the workflow necessary for the generation and analysis of 3D differentiated SIMS cells. SIMS cells are cultured in a 3D matrigel/collagen matrix over a period of 7-9 days. Matrigel is a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma, which is rich in several growth factors and extracellular matrix proteins, such as laminin-1, collagen IV, and heparin sulfate proteoglycans (Patel et al., 2016). Matrigel has been shown to induce differentiation of stem/progenitor cells and outgrowth of already differentiated cell types by causing polarization of cells embedded in or on top of it. For SIMS grown in this 3D matrix, organoid formation post single cell culture is apparent around day 5 of culture. These organoids are also stained either via cryosectioning or in 3D to analyze for differentiation markers and cell populations. Alterations, such as cell transfections, transductions and/or media changes, to this condition can easily be made to address specific research questions (Athwal et al., 2019). The media components were adjusted using literature from both existing salivary and mammary gland differentiation protocols, which deemed necessary to induce differentiation of the SIMS cells in vitro. Here we show the robust use of this cell line to address specific research questions without having to rely on primary salivary gland cells.