Investigations into the metabolism of two-dimensional colony and suspended microcarrier cultures of human embryonic stem cells in serum-free media.

Metabolic studies of human embryonic stem cells (hESCs) can provide important information for stem cell bioprocessing. To this end, we have examined growth and metabolism of hESCs in both traditional 2-dimensional (2D) colony cultures and 3-dimensional microcarrier cultures using a conditioned medium and 3 serum-free media. The 2D colony cultures plateaued at cell densities of 1.1-1.5 × 10⁶ cells/mL at day 6 due to surface limitation. Microcarrier cultures achieved 1.5-2 × 10⁶ cells/mL on days 8-10 before reaching a plateau; this growth arrest was not due to surface limitation, but probably due to metabolic limitations. Metabolic analysis of the cultures showed that amino acids (including glutamine) and glucose are in excess and are not limiting cell growth; on the other hand, the high levels of waste products (25 mM lactate and 0.8 mM ammonium) and low pH (6.6) obtained at the last stages of cell propagation could be the causes for growth arrest. hESCs cultured in media supplemented with lactate (up to 28 mM) showed reduced cell growth, whereas ammonium (up to 5 mM) had no effect. Lactate and, to a lesser extent, ammonia affected pluripotency as reflected by the decreasing population of cells expressing pluripotent marker TRA-1-60. Feeding hESC cultures with low concentrations of glucose resulted in lower lactate levels (∼10%) and a higher pH level of 6.7, which leads to a 40% increase in cell density. We conclude that the high lactate levels and the low pH during the last stages of high-density hESC culture may limit cell growth and affect pluripotency. To overcome this limitation, a controlled feed of low levels of glucose and online control of pH can be used.

Duke Scholars

Author Seog Oh Physics