We describe the design, fabrication, and performance of a bioreactor that enables both morphogenesis of 3D tissue structures under continuous perfusion and repeated in situ observation by light microscopy. Three-dimensional scaffolds were created by deep reactive ion etching of silicon wafers to create an array of channels (through-holes) with cell-adhesive walls. Scaffolds were combined with a cell-retaining filter and support in a reactor housing designed to deliver a continuous perfusate across the top of the array and through the 3D tissue mass in each channel. Reactor dimensions were constructed so that perfusate flow rates meet estimated values of cellular oxygen demands while providing fluid shear stress at or below a physiological range (<2 dyne cm(2)), as determined by comparison of numerical models of reactor fluid flow patterns to literature values of physiological shear stresses. We studied the behavior of primary rat hepatocytes seeded into the reactors and cultured for up to 2 weeks, and found that cells seeded into the channels rearranged extensively to form tissue like structures and remained viable throughout the culture period. We further observed that preaggregation of the cells into spheroidal structures prior to seeding improved the morphogenesis of tissue structure and maintenance of viability. We also demonstrate repeated in situ imaging of tissue structure and function using two-photon microscopy.

The oxidation of propylene preadsorbed on the Pt(111) surface has been characterized in oxygen pressures up to 0.02 Torr using fluorescence yield near-edge spectroscopy (FYNES) and temperature-programmed fluorescence yield near-edge spectroscopy (TP-FYNES) above the carbon K edge. During oxidation of adsorbed propylene, a stable intermediate was observed and characterized using these soft X-ray methods. A general in situ method for determining the stoichiometry of carbon-containing reaction intermediate species has been developed and demonstrated for the first time. Total carbon concentration measured during temperature-programmed reaction studies clearly indicates a reaction intermediate is formed in the 300 K temperature range with a surface concentration of 0.55 × 1015 carbon atoms/cm2. By comparing the intensity of the C−H σ* resonance at the magic angle with the intensity in the carbon continuum, the stoichiometry of this intermediate can be determined unambiguously. Based on calibration with molecular propylene (C3H6) and propylidyne (C3H5), the intermediate has a C3Η5 stoichiometry for oxygen pressures up to 0.02 Torr. A set of normal and glancing angle FYNES spectra above the carbon K edge was used to characterize the bonding and structure of this intermediate. Spectra of known coverages of adsorbed propylene and propylidyne served as standards. The spectra of di-σ propylene, propylidyne, and the intermediate were curve fit as a group with consistent energies and widths of all primary features. Based on this procedure, the intermediate is 1,1,2-tri-σ 1-methylvinyl. The stoichiometry and temperature stability range of the 1-methylvinyl intermediate formed in oxygen pressures up to 0.02 Torr is identical with the stoichiometry and stability of the same intermediate formed during oxidation of preadsorbed propylene by excess coadsorbed atomic oxygen.

In most laboratory-scale mammalian cell cultures, the primary mode of oxygen delivery to cultured cells is by passive diffusion through a thin layer of culture medium, and the height of culture medium chosen may therefore have a significant effect on the phenotype of oxygen-sensitive cell types. Many of the liver functions performed by hepatocytes are thought to be regulated into zones by the local oxygen concentration; of particular interest to in vitro toxicologists, the cytochrome P450 family of detoxification enzymes is known to be preferentially expressed by hepatocytes at low (perivenous) oxygen concentrations. Using an array of different medium heights in a 12-well plate format, we show that the height of culture medium has a significant effect on cytochrome P450 1A1 detoxification activity, glucose metabolism, and cell morphology of HepG2 hepatocellular carcinoma cultures. In particular, cytochrome P450 activity exhibits a maximum at medium heights corresponding to perivenous oxygen concentrations. This work demonstrates that optimizing cell culture performance is not always the same as maximizing oxygen delivery.

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