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

Abstract

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 3-D culture systems provide microenvironmental cues that affect hESC pluripotency and differentiation fates, but little is known about how 3-D culture affects cell expansion. Here we have used a 3-D microwell array to model the differences in hESC growth kinetics and metabolism in 2-D vs. 3-D cultures. Our results demonstrated that 3-D 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 2-D and 3-D cultures. Elucidating the effects of 3-D culture on growth and metabolism of hESCs may facilitate efforts for developing integrated, scalable cell expansion and differentiation processes with these cells.

High-Throughput Single-Cell Kinetics of Virus Infections in the Presence of Defective Interfering Particles

“…Common antiviral factors include interferons (IFNs), and IFN-mediated responses can be triggered by DIPs (63, 64); however, the host BHK cells used here lack such responses (65,66). Alternatively, confinement of cells to microwells can reduce the capacity of cells to grow, as reflected by longer G 1 and shorter G 2 /M phases of the cell cycle (67), conditions that are consistent with lower average productivities of VSV from BHK cells (51). Thus, the lower virus yields associated with isolated coinfected cells (Fig.…”

Enzymatically degradable poly(ethylene glycol) hydrogels for the 3D culture and release of human embryonic stem cell derived pancreatic precursor cell aggregates

“…While suspension cultures support cellular aggregates, they do not afford tight control over aggregate size and are not able to easily provide ECM cues. Alternative strategies such as microwells have been used to control aggregate size [15], but long-term culture and incorporating ECM molecules are more challenging. Given the clinical potential of hESC derived pancreatic precursors [16] and the recent evidence demonstrating for the first time the ability to achieve hESC derived insulin producing cells [17,18], there is a need to establish improved 3D culture platforms for hESC derived pancreatic precursors.…”

Advances in applications of metabolomics in pluripotent stem cell research

“…In addition to studying the effects of drugs, metabolomics has also been used to understand the effect of medium components [42,43••], physiological and atmospheric oxygen concentrations [44,45•], 2D vs. 3D culture [46], and enzymatic passaging [47] on stem cell metabolism. Batch-to-batch variation in media can negatively impact the reproducibility of stem cell culture and differentiation.…”

“…Azarin et al [46] studied the impact of 2D vs. 3D culture of hESCs on their cell cycle and metabolism and observed negligible changes in lactate-glucose ratios due to 3D culture, although they did report that 100 μm diameter hESC colonies contained higher lactate-glucose ratios than larger 3D colonies; the smaller colonies also exhibited less spontaneous differentiation than larger colonies [48], perhaps indicating a relationship between lactate-glucose ratio and hPSC self-renewal. Similarly, different methods of enzymatic passaging of hPSCs resulted in reduction of lipogenesis and glucose utilization in the central carbon metabolism as compared to non-enzymatic dissociation [47] (Table 1).…”