Assessment of the Culture Requirements for Optimal In Vitro Growth and Survival of Multiple Myeloma (MM) Cells.
Authors: Zlei Mihaela , Egert Sabine , Bayer Christine , Wider Dagmar , Schüler Julia , Engelhardt Monika 
Published: October 2019

Abstract
Since MM expands preferentially in the bone marrow (BM) and the close myeloma and stroma interaction is well recognized, we analyzed whether various soluble factors and different stroma can enhance in vitro MM growth. The expansion and protective function of various growth factors (A. 10ng/ml of each IL-6, VEGF, IGF, +/−super (s) IL-6; B. combination A. plus 10ng/ml of each VEGF, HGF, and IL13, and C. 10ng/ml IL-6, IGF, SDF1, Galectin and IL-1) and different stroma (BM stroma from healthy donors [BMSCs], a mouse stroma line [M210B4], and osteoclasts [OC]) were used for MM cell lines (CL: L363, U266, RPMI 8226). Cell number, viability, percentage and number of CD138+ cells were assessed at day 3 and 6 after culture. The most beneficial culture settings were used to establish their relevance for CD138+ enriched BM cells from MM patients (pts), propagated for up to 14 days in culture. MM cell growth proved to be induced through a cell contact mediated mechanism, this being cytokine and stroma dependent. The most beneficial cytokine combination for CL survival and proliferation was combination B, which could substitute the stroma support. Compared to the control, consisting of culture medium only (RPMI 1640+10%FCS), we detected a median 3.1- (combination B) vs. 1.9-fold increase (BMSCs) at day 3, and 1.24- vs. 1.2-fold increase in cell numbers at day 6. BMSCs proved to be most supportive, especially when used together with combination B (1.8- vs. 1.2-fold increase with BMSCs used alone on day 6 of culture), suggesting that BMSCs provide growth factors acting synergistically with IL-6+VEGF+IGF+sIL-6. The cell viability, albeit not cell expansion potential, was best preserved by OC. With proliferation of myeloma CL, a decrease in the percentage of CD138+ cells was observed, consistent with the reported phenotypic shift of plasma cells (PC) from CD138+ to CD138− during cytokine stimulation. Especially OC favoured the expansion of CD138- cells, suggesting that MM clones require defined conditions for their growth and survival. Culture conditions B and C, with or without BMSCs, could sustain primary BM cells from MM pts for up to 14 days. With the inherent variation of different MM pt samples, the combination of 5 factors (C) was more beneficial for these cells. An additional support for cell viability was provided by BMSCs, especially with longer culture periods (beyond 5 days). The different growth and survival requirements for CL seemed to also apply for CD138+ cells from MM pts, which demonstrated to be remarkably heterogeneous as determined by FACS analysis. We identified at least four different PC populations which coexisted before culture in the same specimen. The phenotype of these cells differed in terms of CD45, CD138, CD38, CD56, CD126, CD221, CD19 and CD28, both in their presence on the cell surface and intensity of expression. Taken together, our data indicate that various culture conditions show substantial differences in their ability to preserve myeloma cells, aimed to reproduce the BM microenvironment where the malignancy develops unconstrained. The availability of in vitro systems will allow testing of novel anti-MM agents, alongside with the development of human MM models in mice.