Hannah K. Heywood

Human mesenchymal stem cells (MSCs) reside under hypoxic conditions in vivo, between 4% and 7% oxygen. Differentiation of MSCs under hypoxic conditions results in inhibited osteogenesis, while chondrogenesis is unaffected. The reasons for these results may be associated with the inherent metabolism of the cells. The present investigation measured the oxygen consumption, glucose consumption and lactate production of MSCs during proliferation and subsequent differentiation towards the osteogenic and chondrogenic lineages. MSCs expanded under normoxia had an oxygen consumption rate of ∼98 fmol/cell/h, 75% of which was azide-sensitive, suggesting that these cells derive a significant proportion of ATP from oxidative phosphorylation in addition to glycolysis. By contrast, MSCs differentiated towards the chondrogenic lineage using pellet culture had significantly reduced oxygen consumption after 24 h in culture, falling to ∼12 fmol/cell/h after 21 days, indicating a shift towards a predominantly glycolytic metabolism. By comparison, MSCs retained an oxygen consumption rate of ∼98 fmol/cell/h over 21 days of osteogenic culture conditions, indicating that these cells had a more oxidative energy metabolism than the chondrogenic cultures. In conclusion, osteogenic and chondrogenic MSC cultures appear to adopt the balance of oxidative phosphorylation and glycolysis reported for the respective mature cell phenotypes. The addition of TGF-β to chondrogenic pellet cultures significantly enhanced glycosaminoglycan accumulation, but caused no significant effect on cellular oxygen consumption. Thus, the differences between the energy metabolism of chondrogenic and osteogenic cultures may be associated with the culture conditions and not necessarily their respective differentiation.

Organ-on-chip (OOC) systems recapitulate key biological processes and responses in vitro exhibited by cells, tissues, and organs in vivo . Accordingly, these models of both health and disease hold great promise for improving fundamental research, drug development, personalized medicine, and testing of pharmaceuticals, food substances, pollutants etc. Cells within the body are exposed to biomechanical stimuli, the nature of which is tissue specific and may change with disease or injury. These biomechanical stimuli regulate cell behavior and can amplify, annul, or even reverse the response to a given biochemical cue or drug candidate. As such, the application of an appropriate physiological or pathological biomechanical environment is essential for the successful recapitulation of in vivo behavior in OOC models. Here we review the current range of commercially available OOC platforms which incorporate active biomechanical stimulation. We highlight recent findings demonstrating the importance of including mechanical stimuli in models used for drug development and outline emerging factors which regulate the cellular response to the biomechanical environment. We explore the incorporation of mechanical stimuli in different organ models and identify areas where further research and development is required. Challenges associated with the integration of mechanics alongside other OOC requirements including scaling to increase throughput and diagnostic imaging are discussed. In summary, compelling evidence demonstrates that the incorporation of biomechanical stimuli in these OOC or microphysiological systems is key to fully replicating in vivo physiology in health and disease.

Mesenchymal stem cells (MSCs) are an attractive cell source for tissue engineering applications due to their multipotentiality and increased expansion potential compared to mature cells. However, the full potential of MSCs for cellular therapies is not realised, due, in part, to premature proliferative senescence and impaired differentiation capacity following expansion under 20% oxygen. Bone marrow MSCs reside under reduced oxygen levels (4%-7% oxygen), thus this study investigates the effects of uninterrupted physiological oxygen tensions (2%, 5%) on MSC expansion and subsequent differentiation. Expansion potential was evaluated from colony formation efficiency, population-doubling rates, and cellular senescence. Colony formation was significantly reduced under 5% oxygen compared to 2% and 20% oxygen. Population-doubling time was initially shorter with 20% oxygen, but subsequently no significant differences in doubling time were detected between the oxygen conditions. MSCs expanded with 20% oxygen contained a greater proportion of senescent cells than those under physiological oxygen levels, indicated by a three to fourfold increase in β-galactosidase staining. This may be related to the approximately twofold enhanced mitochondrial oxygen consumption under this culture condition. Chondrogenic differentiation was achieved following expansion at each oxygen condition. However, osteogenesis was only achieved for cells expanded and differentiated at 20% oxygen, indicated by alkaline phosphatase activity and alizarin red staining. These studies demonstrate that uninterrupted hypoxia may enhance long-term MSC expansion, but results in a population with impaired osteogenic differentiation potential. Thus, novel differentiation conditions are required to enable differentiation to nonchondrogenic lineages using hypoxia-cultured MSCs.

The long-term success of any cellular construct used for cartilage tissue engineering is dependent on the maintenance of cell viability throughout the construct thickness. Furthermore, the cells must continue to be metabolically active in order to synthesize a mechanically functional extracellular matrix (ECM). In the present study, a live-dead staining technique and systematic profiling procedure enabled the spatial and temporal distribution of chondrocyte viability to be characterized within 4-mm-thick alginate scaffolds. ECM distribution after 14 days of culture is described both biochemically and histologically and the mechanical functionality of the constructs was assessed by an unconfined compression test. Parameters investigated included alginate permeability, cell-seeding density, and volume of culture medium. Nonhomogeneity of cell and matrix distribution was evident, with greater densities of both parameters in the periphery of the constructs. The culture time preceding central viability loss was inversely related to cell density but relatively independent of scaffold density. However, homogeneity could be attained with increasing medium volume, as evidenced with cell and matrix distribution for cultures in 6.4 mL of medium per 10(6) cells. Moreover, the mechanical properties of the construct were enhanced by culture in increasing volumes of medium. This work indicates that cellular utilization determines the nonhomogeneous nature of cartilage formation in three-dimensional constructs and presents a guide to nonlimiting medium volumes for static culture conditions.

The glycolytic response of hypoxic cells is primarily mediated by the hypoxia inducible factor alpha (HIF-1α) but even in the presence of abundant oxygen tumours typically show high rates of glycolysis. Higher levels of HIF-1α in tumours are associated with a poorer prognosis and up-regulation of markers of epithelial mesenchymal transition (EMT) due to HIF-1α actions. We have recently shown that EMT occurs within the CD44(high) cancer stem cell (CSC) fraction and that epithelial and EMT CSCs are distinguished by high and low ESA expression, respectively. We here show that hypoxia induces a marked shift of the CSC fraction towards EMT leading to altered cell morphology, an increased proportion of CD44(high)/ESA(low) cells, patterns of gene expression typical of EMT, and enhanced sphere-forming ability. The size of EMT fractions returned to control levels in normoxia indicating a reversible process. Surprisingly, however, even under normoxic conditions a fraction of EMT CSCs was present and maintained high levels of HIF-1α, apparently due to actions of cytokines such as TNFα. Functionally, this EMT CSC fraction showed decreased mitochondrial mass and membrane potential, consumed far less oxygen per cell, and produced markedly reduced levels of reactive oxygen species (ROS). These differences in the patterns of oxygen metabolism of sub-fractions of tumour cells provide an explanation for the general therapeutic resistance of CSCs and for the even greater resistance of EMT CSCs. They also identify potential mechanisms for manipulation of CSCs.