Influences of varying oxygen, glucose, and lactate concentrations on the osteogenic development of adipose derived mesenchymal stem cells

Abstract

The aim of this work is to investigate the impact of varying cell culture conditions on the osteogenic differentiation of adMSCs. Glucose and oxygen supply are two parameters which are known for their strong impact on cell behavior. Therefore, the effect of glucose and atmospheric oxygen concentration are examined in this study individually and in combination. Cells are cultivated over a period of 12 days under either high (21%) or low (5%) atmospheric oxygen concentrations and in osteogenic differentiation media with high (4%) or low (1%) glucose concentration. Medium change is carried out in a batch-process approach: A complete medium change is performed every three days to replenish glucose as well as other nutrients and growth factors and to remove lactate and other waste products. Daily measurements of glucose and lactate are performed to compare metabolic activity of the cells under the different cultivation conditions. Special focus is put on discriminating between the effects of glucose concentration and the metabolic changes in response to rising lactate levels. Differentiation is monitored by staining for ECM mineralization: Alizarin red and calcein staining are performed to detect calcification, while phosphate deposition is visualized by von Kossa staining. Cell load and cell distribution are shown by DAPI fluorescence staining of cell nuclei. Results of the study show glucose restriction in the presence of the low- glucose medium. Based on staining results for normoxia experiencing adMSCs, low- and high-glucose media lead to comparable results concerning the osteogenic differentiation. Regarding oxygen availability normoxic (21% oxygen) conditions result in lower glucose consumption rates and a stronger and earlier ECM ossification compared to atmospheric conditions of 5% oxygen. Alizarin red staining significantly detects ECM calcification as early as on differentiation day 7 when cultured under normoxic conditions. This behavior is observed for young (passage zero) adMSCs and passage number of the donor cells could be a crucial impact on the cell response to normoxic and hypoxic conditions. Generally, earlier and stronger mineralization is observed close to the border of the cell culture vessel. The study also shows a correlation between low lactate yield ratios and strong osteogenic differentiation.