The synergistic effect of nanotopography and sustained dual release of hydrophobic and hydrophilic neurotrophic factors on human mesenchymal stem cell neuronal lineage commitment.

A combination of nanotopography and controlled release is a potential platform for neuronal tissue engineering applications. Previous studies showed that combining both physical and chemical guidance was more effective than individual cues in the directional promotion of neurite outgrowth. Nanotopography can direct human mesenchymal stem cells (hMSCs) into neuronal lineage, while controlled release of neurotrophic factors can deliver temporally controlled biochemical signals. Hypothesizing that the synergistic effect will enhance neuronal lineage commitment of hMSCs, a fabrication method for multiple neurotrophic factors delivery from a single nanopatterned (350 nm gratings), poly-ɛ-caprolactone (PCL) film was developed and evaluated. Our results showed a synergistic effect on hMSC differentiation cultured on substrates with both nanotopographical and biochemical cues. The protein/drug encapsulation into PCL nanopatterned films was first optimized using a hydrophilic model protein, bovine serum albumin. The hydrophobic retinoic acid (RA) molecule was directly incorporated into PCL films. To achieve sustained release, hydrophilic nerve growth factor (NGF) was first encapsulated within polyelectrolyte complexation fibers before they were embedded within the nanopatterned PCL film. Our results showed that nanotopography on the fabricated polymer films remained intact, while release of bioactive RA and NGF was sustained over a period of 3 weeks. Under the combinatorial effect of physical and biochemical cues, we observed an enhanced upregulation of neuronal genes such as microtubule-associated protein 2 (MAP2) and neurofilament light (NFL) as compared with sustained delivery of individual cues and bolus delivery. Quantitative polymerase chain reaction analysis showed that MAP2 and NFL gene upregulation in hMSCs was most pronounced on the nanogratings with sustained release of both RA and NGF. The fabricated platforms supported the sustained delivery of multiple neurotrophins, including both hydrophobic and hydrophilic therapeutic agents, while providing surface patterning versatility for application in neural regeneration and tissue engineering.

Computational fluid model incorporating liver metabolic activities in perfusion bioreactor.

The importance of in vitro hepatotoxicity testing during early stages of drug development in the pharmaceutical industry demands effective bioreactor models with optimized conditions. While perfusion bioreactors have been proven to enhance mass transfer and liver specific functions over a long period of culture, the flow-induced shear stress has less desirable effects on the hepatocytes liver-specific functions. In this paper, a two-dimensional human liver hepatocellular carcinoma (HepG2) cell culture flow model, under a specified flow rate of 0.03 mL/min, was investigated. Besides computing the distribution of shear stresses acting on the surface of the cell culture, our numerical model also investigated the cell culture metabolic functions such as the oxygen consumption, glucose consumption, glutamine consumption, and ammonia production to provide a fuller analysis of the interaction among the various metabolites within the cell culture. The computed albumin production of our 2D flow model was verified by the experimental HepG2 culture results obtained over 3 days of culture. The results showed good agreement between our experimental data and numerical predictions with corresponding cumulative albumin production of 2.9 × 10(-5) and 3.0 × 10(-5) mol/m(3) , respectively. The results are of importance in making rational design choices for development of future bioreactors with more complex geometries.

Computational fluid modeling and performance analysis of a bidirectional rotating perfusion culture system.

A myriad of bioreactor configurations have been investigated as extracorporeal medical support systems for temporary replacement of vital organ functions. In recent years, studies have demonstrated that the rotating bioreactors have the potential to be utilized as bioartificial liver assist devices (BLADs) owing to their advantage of ease of scalability of cell-culture volume. However, the fluid movement in the rotating chamber will expose the suspended cells to unwanted flow structures with abnormally high shear conditions that may result in poor cell stability and in turn lower the efficacy of the bioreactor system. In this study, we compared the hydrodynamic performance of our modified rotating bioreactor design with that of an existing rotating bioreactor design. Computational fluid dynamic analysis coupled with experimental results were employed in the optimization process for the development of the modified bioreactor design. Our simulation results showed that the modified bioreactor had lower fluid induced shear stresses and more uniform flow conditions within its rotating chamber than the conventional design. Experimental results revealed that the cells within the modified bioreactor also exhibited better cell-carrier attachment, higher metabolic activity, and cell viability compared to those in the conventional design. In conclusion, this study was able to provide important insights into the flow physics within the rotating bioreactors, and help enhanced the hydrodynamic performance of an existing rotating bioreactor for BLAD applications.

A thin-walled polydimethylsiloxane bioreactor for high-density hepatocyte sandwich culture.

In vitro drug testing requires long-term maintenance of hepatocyte liver specific functions. Hepatocytes cultured at a higher seeding density in a sandwich configuration exhibit an increased level of liver specific functions when compared to low density cultures due to the better cell to cell contacts that promote long term maintenance of polarity and liver specific functions. However, culturing hepatocytes at high seeding densities in a standard 24-well plate poses problems in terms of the mass transport of nutrients and oxygen to the cells. In view of this drawback, we have developed a polydimethylsiloxane (PDMS) bioreactor that was able to maintain the long-term liver specific functions of a hepatocyte sandwich culture at a high seeding density. The bioreactor was fabricated with PDMS, an oxygen permeable material, which allowed direct oxygenation and perfusion to take place simultaneously. The mass transport of oxygen and the level of shear stress acting on the cells were analyzed by computational fluid dynamics (CFD). The combination of both direct oxygenation and perfusion has a synergistic effect on the liver specific function of a high density hepatocyte sandwich culture over a period of 9 days.