This study aims to quantify the metabolic flux in endothelial progenitor cells in order to characterize the metabolic changes occurring during in vitro culturing. It has previously been shown that the cellular microenvironment is a major determinant in the progression of viable and functional cells. Thus we here investigate the metabolic profile of two cell setups, 3D scaffolds and suspension, to underline the effect of the cultivation method used to expand cell populations. Introduction Endothelial progenitor cells (EPCs) represent a heterogeneous cell population, believed to be involved in vasculogenesis after ischemic diseases[1]. With the purpose of enhancing endothelial repair, EPCs could have a potential for future cell therapies, and this potential is widely investigated[2]. Due to the low amount of EPCs in the peripheral circulating blood they have to be expanded in vitro before administration to recipients and the effects of in vitro culturing is still an under-evaluated field with little knowledge of how the cells change in both phenotype and morphology[3]. EPCs culture in 3D scaffolds or in suspension are transferred to the bioreactor system and placed in a 9.4T horizontal magnet .The lactate-to-pyruvate ratio was measured by injection of hyperpolarized [1-¹³C] pyruvate EPCs cultures subjected to either 3D scaffolds or cell suspension. Additionally lactate dehydrogenase (LDH) and pyruvate dehydrogenase (PDH) activity was measured with commercial activity assays on cells from both experiment setups. qPCR was conducted to address the expression rate of the enzymes PDH and LDH. To evaluate the presence and the number of cells housing the scaffold, fluorescents microscopy of the scaffolds and measurements of DNA concentration was performed.Seeding and housing of EPCs in 3D scaffolds were confirmed by fluorescence microscopy (Figure 1) and measurements of DNA concentration. The DNA concentration of each scaffold reached 4580ng/ml compared to a DNA concentration of 3900 ng/ml for 1 million suspension cells. Each scaffold is housing app. 1.5 mio cells, and during hyperpolarization measurements a total of 10 mio cells was used. The lactate-to-pyruvate ratio (3±0,27) was significantly higher in cell suspensions compared to the cells adhered to scaffolds (0.5±0,80)(p<0,0001)(Figure 2A and b). This increase stayed constant during the culturing period of approximately 25 days. The LDH activity increased from 0,23±0,01 milliunits/mL in the suspension cultures to 3,2±1,2 milliunits/mL in the scaffolds. The current setup was too insensitive to measure the PDH activity in the commercial activity assay. The mRNA expression of PDH was 3 fold higher in suspension cells compared to scaffold culturing, and the initial experiments for LDH expression showed general low expression in both suspension and scaffold setups. Interestingly, we find that the pyruvate to lactate conversion was elevated in the endothelial progenitor cells when measurements were done on cells in suspension compared to cells adhered to scaffolds. The activity assay further underlines that the LDH is more active in the suspension cells and the low activity signal from PDH suggests that the enzyme is inhibited. The increased PDH expression could be a means of the cells compensating for the environmental struggle suspension culturing infuses. Further experiments to investigate the cell viability in the two setups will be conducted to underline this hypothesis. We have here demonstrated the potential of monitoring cell senescence using hyperpolarized MRS as well as indicated metabolomic changes of progenitor cells when exposed to suspension as this is non optimal conditions for adherent cells.