Effects of 3D microwell culture on growth kinetics and metabolism of human embryonic stem cells

Human embryonic stem cells (hESCs) hold potential in the field of tissue engineering, given their capacity for both limitless self-renewal and differentiation to any adult cell type. However, several limitations, including the ability to expand undifferentiated cells and efficiently direct differentiation at scales needed for commercial cell production, prevent realizing the potential of hESCs in tissue engineering. Numerous studies have illustrated that three-dimensional (3D) culture systems provide microenvironmental cues that affect hESC pluripotency and differentiation fates, but little is known about how 3D culture affects cell expansion. Here, we have used a 3D microwell array to model the differences in hESC growth kinetics and metabolism in two-dimensional (2D) versus 3D cultures. Our results demonstrated that 3D microwell culture reduced hESC size and proliferative capacity, and impacted cell cycle dynamics, lengthening the G1 phase and shortening the G2/M phase of the cell cycle. However, glucose and lactate metabolism were similar in 2D and 3D cultures. Elucidating the effects of 3D culture on growth and metabolism of hESCs may facilitate efforts for developing integrated, scalable cell expansion and differentiation processes with these cells.