Development of a 3D Colture Platform for Multiple Myeloma Tumor Cells

The aim of this work is to contribute to the development of a 3D culture platform for Multiple Myeloma tumor cells in order to enable practical testing of antitumoral drugs. To achieve this, it is essential to replicate in vitro the interactions that Multiple Myeloma cells (MMCs) maintain in vivo with other cells of the tumor microenvironment, in particular mesenchymal stem cells (MSCs), and with proteins and polysaccharides of the bone marrow extracellular matrix. In order to do this, the cells must be cultured in a three-dimensional environment containing all the components of the real tumor microenvironment. At the Centre of Biomaterials and Tissue Engineering (CBIT), a model based on microgel is being developed. It consists in a suspension of microspheres in a liquid culture medium in which the MMCs will also be suspended. The culture conditions play a crucial role in preserving the viability and proliferation of these cells. However, due to the disease's heterogeneity, the behavior of each of the accessible MM cell lines can vary greatly. In this work, with the help of microfluidics, we have made progress in the production and characterization of biomimetic microgels, which are based on alginate microspheres. The microspheres are approximately 150 μm in diameter and are covered in a multilayer of polyelectrolytes. The first layers of alginate and poly-L-lysine ensure the stability of the microsphere and are followed by a sequence of layers that present the polysaccharide of interest on the microsphere's surface. The three-dimensional environment of the cells consists of a mixture of microspheres functionalized with collagen, hyaluronic acid, heparin and chondroitin sulfate and suspended in Roswell Park Memorial Institute (RPMI) culture medium to which the cells are added. The presence of these extracellular matrix components on the surface of the microspheres has been characterized. MMCs and supporting microspheres are deposited on conical agarose wells designed and produced by us. These wells are permeable to water and water-soluble substances. Co-culture with mesenchymal stem cells (MSCs) is performed by producing pellets of agglomerated cells with adherent microparticles for them. These pellets are added either inside or outside the wells to study the effect of direct or indirect contact between MSCs and MMCs. We first conducted a study on the effect of the frequency of changes of the liquid culture medium and other parameters of the culture protocol that probably determine the content in the medium of nutrients, as well as cytokines and other factors secreted by the cells. We tested the two most common glucose concentrations (2 g/L and 4.5 g/L) and found that changing the medium with the lowest concentration of glucose completely every two days yielded the best results. Afterward, we tried to test the viability of the MM1.S cell line on the platform. Previous results of the response of cells from the RPMI8226 line were available, showing that the microgel favored cell proliferation and that there was little difference between direct and indirect co-culture. Very different behavior was found in the cells of MM1.S line compared to those of RPMI8226 line in terms of much lower proliferation both when cultured in the 3D environment created by the microgel and when cultured in suspension in the absence of microspheres. Finally, cell viability studies were carried out on the platform by subjecting the cells to treatment with Dexamethasone.