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Abstract  A tissue-mimetic culture system (TMCS) in which cells are sandwiched between two glass slides provides an ideal microenvironment for studying the effects of oxygen and nutrient gradients on cells in culture. A mathematical model was utilized to predict the time course of the development of oxygen and glucose concentration gradients within the TMCS. Oxygen and glucose consumption rates of mouse embryonic stem cells were measured as parameters for the model. The model predicts oxygen and glucose concentration profiles directly using a single experimentally controlled variable, the seeding density of cells within the system. The model predicts that the time required for the gradients to reach steady state is inversely related to the cell density, and the penetration depth of the gradients into the TMCS is inversely related to the square root of the cell density. Experimental oxygen concentration measurements were performed at a cell density of 9.1×106 cells cm−3, and the gradient was found to develop to a steady-state profile within 20 min and penetrate approximately 2 mm into the TMCS, consistent with the theoretical predictions. This model and the TMCS provide useful tools for investigating the effect of the metabolic microenvironment on cells in culture. [ABSTRACT FROM AUTHOR]