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.

Tuberculosis is the number one cause of death due to infectious disease in the world today. Understanding the dynamics of the immune response is crucial to elaborating differences between individuals who contain infection vs those who suffer active disease. Key cells in an adaptive immune response to intracellular pathogens include CD8+ T cells. Once stimulated, these cells provide a number of different effector functions, each aimed at clearing or containing the pathogen. To explore the role of CD8+ T cells in an integrative way, we synthesize both published and unpublished data to build and test a mathematical model of the immune response to Mycobacterium tuberculosis in the lung. The model is then used to perform a series of simulations mimicking experimental situations. Selective deletion of CD8+ T cell subsets suggests a differential contribution for CD8+ T cell effectors that are cytotoxic as compared with those that produce IFN-γ. We also determined the minimum levels of effector memory cells of each T cell subset (CD4+ and CD8+) in providing effective protection following vaccination.

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A fluorescence lifetime imaging microscopy (FLIM) method was developed and applied to investigate metabolic function in living human normal esophageal (HET-1) and Barrett's adenocarcinoma (SEG-1) cells. In FLIM, image contrast is based on fluorophore excited state lifetimes, which reflect local biochemistry and molecular activity. Unique FLIM system attributes, including variable ultrafast time gating (>/= 200 ps), wide spectral tunability (337.1 - 960 nm), large temporal dynamic range (>/= 600 ps), and short data acquisition and processing times (15 s), enabled the study of two key molecules consumed at the termini of the oxidative phosphorylation pathway, NADH and oxygen, in living cells under controlled and calibrated environmental conditions. NADH is an endogenous cellular fluorophore detectable in living human tissues that has been shown to be a quantitative biomarker of dysplasia in the esophagus. Lifetime calibration of an oxygen-sensitive, ruthenium-based cellular stain enabled in vivo oxygen level measurements with a resolution of 8 muM over the entire physiological range (1 - 300 muM). Starkly higher intracellular oxygen and NADH levels in living SEG-1 vs. HET-1 cells were detected by FLIM and attributed to altered metabolic pathways in malignant cells.

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...

The immune response to Mycobacterium tuberculosis (Mtb) infection is complex. Experimental evidence has revealed that tumor necrosis factor (TNF) plays a major role in host defense against Mtb in both active and latent phases of infection. TNF-neutralizing drugs used to treat inflammatory disorders have been reported to increase the risk of tuberculosis (TB), in accordance with animal studies. The present study takes a computational approach toward characterizing the role of TNF in protection against the tubercle bacillus in both active and latent infection. We extend our previous mathematical models to investigate the roles and production of soluble (sTNF) and transmembrane TNF (tmTNF). We analyze effects of anti-TNF therapy in virtual clinical trials (VCTs) by simulating two of the most commonly used therapies, anti-TNF antibody and TNF receptor fusion, predicting mechanisms that explain observed differences in TB reactivation rates. The major findings from this study are that bioavailability of TNF following anti-TNF therapy is the primary factor for causing reactivation of latent infection and that sTNF—even at very low levels—is essential for control of infection. Using a mathematical model, it is possible to distinguish mechanisms of action of the anti-TNF treatments and gain insights into the role of TNF in TB control and pathology. Our study suggests that a TNF-modulating agent could be developed that could balance the requirement for reduction of inflammation with the necessity to maintain resistance to infection and microbial diseases. Alternatively, the dose and timing of anti-TNF therapy could be modified. Anti-TNF therapy will likely lead to numerous incidents of primary TB if used in areas where exposure is likely.

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