Lumox

Gas transfer was evaluated in a custom-made modular bioreactor system (depicted in Figure 1) that ensures a laminar flow and a well-defined wall shear stress in the range of physiological values. The reactor itself consists of two chambers, a gas-carrying chamber, and a test fluid-filled chamber, which are separated from one another by a gas permeable membrane. The employed membrane is a nonporous fluorocarbon membrane (lumox®, Sarstedt) with a thickness of 25 μm that allows for cultivation of adherent cells surface treatment (not specified) carried out by the manufacturer.
In terms of a laminar and steady flow of an incompressible frictional fluid between parallel plates, the wall shear stress (WSS) in the test fluid-filled chamber is given by the following [25 (link)]: $\begin{array}{c}{\tau }_{\mathrm{0}}=\frac{\mathrm{3}·\eta ·\dot{V}}{\mathrm{2}·b·h^{\mathrm{2}}}.\end{array}$
Here, τ₀ is the shear stress at the wall, η = 0.693 mPa s is the dynamic viscosity of culture medium at 37°C, $\dot{V}$ is the flow rate, b is the width, and h is half the distance between the plates. For this chamber, the mean retention time t_t is given by the quotient of the reactor volume V_R to the flow rate $\dot{V}$ ; hence $\begin{array}{c}{t}_t=\frac{V_R}{\dot{V}}.\end{array}$ The parameters for this calculation are given in Table 1.

The intestinal epithelial cell line T84 (CCL-248; ATCC, Molsheim, France) was maintained in an atmosphere containing 5% CO₂ at 37 °C in the culture medium recommended by ATCC (Molsheim, France). T84 cells were cultivated using conventional polystyrene dishes or special gas-permeable dishes with a hydrophilic tissue culture-treated bottom membrane (Lumox; Sarstedt, Marnay, France) as previously used [43 (link)].

Aldehyde/alcohol dehydrogenase activity was measured indirectly by evaluating oxidation or reduction of NAD(P)(H) photometrically at 340 nm in triplicate in 96-well plates (lumox^®, Sarstedt, Nümbrecht, Germany) using an MRX ELISA reader (Dynex Technologies, Chantilly, VA, USA).
The assays were performed depending on the pH in a 100 mM glycine buffer (pH 8, 9), 100 mM Tris buffer (pH 7, 7.4, 6) at a total volume of 200 µL containing 4 µg recEhADH3B^b, 0.1–100 mM substrate, and 0.7 mM NAD(P)H/NAD(P). The amount of enzyme which is needed to catalyze the oxidation or reduction of 1 μmol of NAD(P)(H) is defined as one unit. The following substrates were tested: Methanol, ethanol, 1-propanol, 2-propanol, butanol, formaldehyde (methanal), acetaldehyde (ethanal), propionaldehyde (propanal) and butyraldehyde (butanal). EDTA (10 mM) was incubated with the enzyme for 1 h on ice. To remove EDTA, the enzyme solution was dialysed against an EDTA-free buffer. Different metal ions at a concentration of 1 mM were added to the dialyzed enzyme solution and incubated for 1 h on ice. Subsequently acetaldehyde-dependent oxidation of NAD(P)H was measured. The metal-free and dialyzed recEhADH3B^b served as negative control. As positive control the NAD(H)-dependent alcohol dehydrogenase from Saccharomyces cerevisiae (Merck, Sigma Aldrich, Darmstadt, Germany) was used.