Comprehensive multiplexed superfusion system enables physiological emulation in cell culture: exemplification by persistent circadian entrainment.

Cells and tissues are routinely cultured in vitro for biological research with findings being extrapolated to their host organ and tissue function. However, most samples are cultured and studied in unphysiological environments, without temporal variation in the biochemical cues that are ubiquitous in vivo. The artificiality of these conditions undermines the predictive value of cell culture studies. We ascribe the prevalence of this suboptimal culture methodology to the lack of practical continuous flow systems that are economical and robust. Here, we design and implement an expandable multiplexed flow system for cell culture superfusion. By expanding on the concept of the planar peristaltic pump, we fabricated a highly compact and multiplexed pump head with up to 48 active pump lines. The pump is incorporated into a custom, open-top superfusion system configured for conventional multi-well culture plates. We then demonstrated the utility of the system for in vitro circadian entrainment using a daily cortisol pulse, generating a sustained circadian amplitude that is essential for physiological emulation and chrono-pharmacological studies. The multiplexed pump is complemented by a package of fluidic interconnection and management methods enabling user-friendly and scalable operation. Collectively, the suite of technologies provides a much-needed improvement in physiological emulation to support the predictive value of in vitro biomedical and biological research.

On-chip surface acoustic wave and micropipette aspiration techniques to assess cell elastic properties.

The cytoskeletal mechanics and cell mechanical properties play an important role in cellular behaviors. In this study, in order to provide comprehensive insights into the relationship between different cytoskeletal components and cellular elastic moduli, we built a phase-modulated surface acoustic wave microfluidic device to measure cellular compressibility and a microfluidic micropipette-aspiration device to measure cellular Young's modulus. The microfluidic devices were validated based on experimental data and computational simulations. The contributions of structural cytoskeletal actin filament and microtubule to cellular compressibility and Young's modulus were examined in MCF-7 cells. The compressibility of MCF-7 cells was increased after microtubule disruption, whereas actin disruption had no effect. In contrast, Young's modulus of MCF-7 cells was reduced after actin disruption but unaffected by microtubule disruption. The actin filaments and microtubules were stained to confirm the structural alteration in cytoskeleton. Our findings suggest the dissimilarity in the structural roles of actin filaments and microtubules in terms of cellular compressibility and Young's modulus. Based on the differences in location and structure, actin filaments mainly contribute to tensile Young's modulus and microtubules mainly contribute to compressibility. In addition, different responses to cytoskeletal alterations between acoustophoresis and micropipette aspiration demonstrated that micropipette aspiration was better at detecting the change from actin cortex, while the response to acoustophoresis was governed by microtubule networks.

Rhythm on a chip: circadian entrainment in vitro is the next frontier in body-on-a chip technology.

Organoids, bioprinted mini-tissues and body-on-a-chip technologies are poised to transform the practice of preclinical pharmacology, with a view to achieving better predictive value. We review the need for further refinement in static and dynamic biomechanical aspects of such microenvironments. Further consideration of the developments required in perfusion systems to enable delivery of an appropriate soluble microenvironment are argued. We place particular emphasis on a major deficiency in these systems, being the absence or aberrant circadian behaviour of cells used in such settings, and consider the technical challenges that are needing to be met in order to achieve rhythm-on-a-chip.

Control of epithelial cell migration and invasion by the IKKß- and CK1α-mediated degradation of RAPGEF2.

Epithelial cell migration is crucial for the development and regeneration of epithelial tissues. Aberrant regulation of epithelial cell migration has a major role in pathological processes such as the development of cancer metastasis and tissue fibrosis. Here, we report that in response to factors that promote cell motility, the Rap guanine exchange factor RAPGEF2 is rapidly phosphorylated by I-kappa-B-kinase-ß and casein kinase-1α and consequently degraded by the proteasome via the SCF(ßTrCP) ubiquitin ligase. Failure to degrade RAPGEF2 in epithelial cells results in sustained activity of Rap1 and inhibition of cell migration induced by HGF, a potent metastatic factor. Furthermore, expression of a degradation-resistant RAPGEF2 mutant greatly suppresses dissemination and metastasis of human breast cancer cells. These findings reveal a molecular mechanism regulating migration and invasion of epithelial cells and establish a key direct link between IKKß and cell motility controlled by Rap-integrin signaling.