Microfluidic bioreactors fabricated from highly gas-permeable poly(dimethylsiloxane) (PDMS) materials have been observed, somewhat unexpectedly, to give rise to heterogeneous long term responses along the length of a perfused mammalian cell culture channel, reminiscent of physiologic tissue zonation that arises at least in part due to oxygen gradients. To develop a more quantitative understanding and enable better control of the physical-chemical mechanisms underlying cell biological events in such PDMS reactors, dissolved oxygen concentrations in the channel system were quantified in real time using fluorescence intensity and lifetime imaging of an oxygen sensitive dye, ruthenium tris(2,2'-dipyridyl) dichloride hexahydrate (RTDP). The data indicate that despite oxygen diffusion through PDMS, uptake of oxygen by cells inside the perfused PDMS microchannels induces an axial oxygen concentration gradient, with lower levels recorded in downstream regions. The oxygen concentration gradient generated by a balance of cellular uptake, convective transport by media flow, and permeation through PDMS in our devices ranged from 0.0003 (mg/l)/mm to 0.7 (mg/l)/mm. The existence of such steep gradients induced by cellular uptake can have important biological consequences. Results are consistent with our mathematical model and give insight into the conditions under which flux of oxygen through PDMS into the microchannels will or will not contribute significantly to oxygen delivery to cells and also provide a design tool to manipulate and control oxygen for cell culture and device engineering. The combination of computerized microfluidics, in situ oxygen sensing, and mathematical models opens new windows for microphysiologic studies utilizing oxygen gradients and low oxygen tensions.

This paper introduces the concept of phase-locking analysis of oscillatory cellular signaling systems to elucidate biochemical circuit architecture. Phase-locking is a physical phenomenon that refers to a response mode in which system output is synchronized to a periodic stimulus; in some instances, the number of responses can be fewer than the number of inputs, indicative of skipped beats. While the observation of phase-locking alone is largely independent of detailed mechanism, we find that the properties of phase-locking are useful for discriminating circuit architectures because they reflect not only the activation but also the recovery characteristics of biochemical circuits. Here, this principle is demonstrated for analysis of a G-protein coupled receptor system, the M3 muscarinic receptor-calcium signaling pathway, using microfluidic-mediated periodic chemical stimulation of the M3 receptor with carbachol and real-time imaging of resulting calcium transients. Using this approach we uncovered the potential importance of basal IP3 production, a finding that has important implications on calcium response fidelity to periodic stimulation. Based upon our analysis, we also negated the notion that the Gq-PLC interaction is switch-like, which has a strong influence upon how extracellular signals are filtered and interpreted downstream. Phase-locking analysis is a new and useful tool for model revision and mechanism elucidation; the method complements conventional genetic and chemical tools for analysis of cellular signaling circuitry and should be broadly applicable to other oscillatory pathways.

Pembrolizumab, a humanized monoclonal antibody against programmed death 1 (PD‐1), has a manageable safety profile and robust clinical activity against advanced malignancies. The lowest effective dose for evaluation in further dose‐ranging studies was identified by developing a translational model from preclinical mouse experiments. A compartmental pharmacokinetic model was combined with a published physiologically based tissue compartment, linked to receptor occupancy as the driver of observed tumor growth inhibition. Human simulations were performed using clinical pharmacokinetic data, literature values, and in vitro parameters for drug distribution and binding. Biological and mathematical uncertainties were included in simulations to generate expectations for dose response. The results demonstrated a minimal increase in efficacy for doses higher than 2 mg/kg. The findings of the translational model were successfully applied to select 2 mg/kg as the lowest dose for dose‐ranging evaluations.

Abstract. For the first time, a fluorescence lifetime calibration method for an oxygen-sensitive dye ruthenium tris͑2 ,2Ј-dipyridyl͒ dichloride hexahydrate ͑RTDP͒ is applied to image oxygen levels in poly͑dimethyl siloxane͒ ͑PDMS͒ bioreactors containing living C2C12 mouse myoblasts. PDMS microsystems are broadly used in bioengineering applications due to their biocompatibility and ease of handling. For these systems, oxygen concentrations are of significance and are likely to play an important role in cell behavior and gene expression. Fluorescence lifetime imaging microscopy ͑FLIM͒ bases image contrast on fluorophore excited state lifetimes, which reflect local biochemistry. Unique attributes of the widefield, timedomain FLIM system include tunable excitation ͑337.1 to 960 nm͒, large temporal dynamic range ͑Ն600 ps͒, high spatial resolution ͑1.4 m͒, calibrated detection ͑0 to 300± 8 M of oxygen͒, and rapid data acquisition and processing times ͑10 s͒. Oxygen levels decrease with increasing cell densities and are consistent with model outcomes obtained by simulating bioreactor oxygen diffusion and cell proliferation. In single bioreactor loops, FLIM detects spatial heterogeneity in oxygen levels with variations as high as 20%. The fluorescence lifetime-based imaging approach we describe avoids intensity-based artifacts ͑in-cluding photobleaching and concentration variations͒ and provides a technique with high spatial discrimination for oxygen monitoring in continuous cell culture systems. Microfluidic devices have promising applications in cellbased assays and microscale tissue engineering, where spatiotemporal conditions are readily manipulated. Recently, poly͑dimethyl siloxane͒-͑PDMS͒-based microfluidic systems have been developed as biocompatible and rapidly prototyped systems for microscale-cell culture. For example, cells could be seeded and cultured successfully under continually perfused conditions to achieve an extracellular fluid-to-cell ͑vol-ume͒ ratio close to the physiological value 1 of 0.5. This small ratio facilitates heterogeneous chemical distribution, which may be critical in specifying cell fate in developing tissues. It is hence of great interest to quantitatively and with minimal perturbation characterize components ͑e.g., mitogens, nutrients, oxygen͒ in microfluidic bioreactors that influence cellular responses.Oxygen in cell cultures influences cell signaling, growth, differentiation, and death.1 PDMS bioreactors are popular due to their high diffusivity of oxygen, which has been repeatedly demonstrated.2 It has been observed, 3 however, that the diffusivity of PDMS can vary, depending on protein adsorption ͑e.g., when cells are cultured͒ or surface modification ͑e.g., plasma oxidization for bioreactors͒. It is hypothesized that this variability in PDMS permeability, along with cellular uptake and culture media perfusion, can affect spatial variations in oxygen within PDMS bioreactors.Optical measurements of oxygen sensitive agents have advantages over more traditional, electrode-based app...