Differential dynamical effects of macromolecular crowding on an intrinsically disordered protein and a globular protein: implications for in-cell NMR spectroscopy.

In-cell NMR provides a valuable means to assess how macromolecules, with concentrations up to 300 g/L in the cytoplasm, affect the structure and dynamics of proteins at atomic resolution. Here an intrinsically disordered protein, alpha-synuclein (alphaSN), and a globular protein, chymotrypsin inhibitor 2 (CI2) were examined by using in-cell NMR. High-resolution in-cell spectra of alphaSN can be obtained, but CI2 leaks from the cell and the remaining intracellular CI2 is not detectable. Even after stabilizing the cells from leakage by using alginate encapsulation, no CI2 signal is detected. From in vitro studies we conclude that this difference in detectability is the result of the differential dynamical response of disordered and ordered proteins to the changes of motion caused by the increased viscosity in cells.

Modeling O2 transport within engineered hepatic devices.

Predicting and improving oxygen transport within bioartificial liver (BAL) devices continues to be an important engineering challenge since oxygen is one of the critical nutrients necessary for maintaining hepatocyte viability and function. Such a computational model would not only help predict outcomes but it would also allow system modifications to be analyzed prior to developing experimental protocols. This would help to facilitate future design improvements while reducing both experimental time and capital resource costs, and is the focus of the current study. Specifically, a computational model of O(2) transport through collagen and microporous collagen ECMs is analyzed for hollow fiber (HF), flat plate (FP), and spheroid BAL designs. By modifying the O(2) boundary conditions, hepatocyte O(2) consumption levels, O(2) permeability of the ECM, and ECM void fractions, O(2) transport predictions are determined for each system as a function of time and distance. Accuracy of the predictive model is confirmed by comparing computational vs. experimental results for the HF BAL system. The model's results indicate that O(2) transport within all three BAL designs can be improved significantly by incorporating the enhancement technique. This technique modifies a diffusion-dominant gel ECM into a porous matrix with diffusive and convective flows that mutually transport O(2) through the ECMs. Although tortuous pathways increase the porous ECM's overall effective length of O(2) travel, the decreased transport resistances of these pathways allow O(2) to permeate more effectively into the ECMs. Furthermore, because the HF design employs convective flow on both its inner and outer ECM surfaces, greater control of O(2) transport through its ECM is predicted, as compared with the single O(2) source inputs of the flat plate and spheroid systems. The importance of this control is evaluated by showing how modifying the O(2) concentration and/or transfer coefficients of the convective flows can affect O(2) transport.

A novel multi-coaxial hollow fiber bioreactor for adherent cell types. Part 1: hydrodynamic studies.

A novel multi-coaxial bioreactor for three-dimensional cultures of adherent cell types, such as liver, is described. It is composed of four tubes of increasing diameter placed one inside the other, creating four spatially isolated compartments. Liver acinar structure and physiological parameters are mimicked by sandwiching cells in the space between the two innermost semi-permeable tubes, or hollows fibers, and creating a radial flow of media from an outer compartment (ECC), through the cell mass compartment, and to an inner compartment (ICC). The outermost compartment is created by gas-permeable tubing, and the housing is used to oxygenate the perfusion media to periportal levels in the ECC. Experiments were performed using distilled water to correlate the radial flow rate (Q(r)) with (1) the pressure drop (DeltaP) between the media compartments that sandwich the cell compartment and (2) the pressure in the cell compartment (P(c)). These results were compared with the theoretical profile calculated based on the hydraulic permeability of the two innermost fibers. Phase-contrast velocity-encoded magnetic resonance imaging was used to visualize directly the axial velocities inside the bioreactor and confirm the assumptions of laminar flow and zero axial velocity at the boundaries of each compartment in the bioreactor. Axial flow rates were calculated from the magnetic resonance imaging results and were similar to the measured axial flow rates for the previously described experiments.