Synthesis and performance of 3D-megaporous structures for enzyme immobilization and protein capture

The preparation of megaporous bodies, with potential applications in biotechnology, was attempted by following several strategies. As a first step, naive and robust scaffolds were produced by polymerization of selected monomers in the presence of a highly soluble cross‐linker agent. Ion‐exchange function was incorporated by particle embedding, direct chemical synthesis, or radiation‐induced grafting. The total ionic capacity of such systems was 1.5 mmol H⁺/g, 1.4 mmol H⁺/g, and 17 mmol H⁺/g, respectively. These values were in agreement with the ability to bind model proteins: observed dynamic binding capacity at 50% breakthrough was ?7.2 mg bovine serum albumin/g, ?7.4 hen egg‐white lysozyme (HEWL) mg/g, and ?108 HEWL mg/g. In the later case, total (static) binding capacity reached 220 mg/g. It was observed that the structure and size of the megapores remained unaffected by the grafting procedure which, however, allowed for the highest protein binding capacity. Lysozyme supported on grafted body showed extensive clarification activity against a Micrococcus lysodekticus suspension in the flow‐through mode, i.e., 90% destruction of suspended microbial cells was obtained with a residence time ≈ 18 min. Both protein capture and biocatalysis applications are conceivable with the 3D‐megaporous materials described in this work. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Formation of bile canaliculi in long-term primary cultures of adult rat hepatocytes on permeable membrane: an ultrastructural study

Adult rat hepatocytes were cultured for 15 days on type I collagen-coated permeable membranes in a hormonally defined Waxman's modified medium supplemented with very low concentrations of insulin, glucagon and dexamethasone. Phase contrast examination showed that 15-day-old cultures still formed a regular monolayer of polygonal cells. In similarly aged cultures, intracellular glycogen was abundant and evenly distributed, while steatosis remained very limited. Scanning and transmission electron microscopy showed that well developed bile canaliculi could be observed on the lateral side of the hepatocyte membrane after 4 days of incubation and persisted for 2 weeks. These canalicular structures probably originated from coalescence of membrane invaginations observed in 1-day-old cultures. Transmission electron microscopy showed that the ultrastructure of the cells was very close to that of normal rat hepatocytes in the intact liver. These results suggest that rat hepatocytes cultured under these experimental conditions are able to develop and maintain tissue-specific cytochemical and morphological properties for at least 15 days.     

Formaldehyde cross-linking of gliadin films: effects on mechanical and water barrier properties

In this study, pioneering results on specific chemical modifications of wheat gluten gliadins and the corresponding impact on mechanical and water barrier properties of derived films are presented. Films were prepared from gliadins chemically treated with formaldehyde and subsequently mixed with different concentrations of glycerol as a plasticizing agent. Water vapor barrier and mechanical properties of the films were evaluated as a function of relative humidity and glycerol concentration. Formaldehyde treatment led to enhanced mechanical properties and, to a lesser extent, improved water barrier of the films, effects which point to the formation of new intermolecular bonds between monomeric gliadins. The occurrence of cross-linking was supported by SDS-PAGE analysis. Cross-linked films maintained their integrity after immersion in water and had similar optical properties to control films. The effect of glycerol and humidity on water vapor permeability and the mechanical properties of films was less acute when proteins were treated with formaldehyde. Thus, chemical treatment of proteins is shown to be a very effective route for optimizing the use of these films in packaging applications.     

Rheology of passive and adhesion-activated neutrophils probed by atomic force microscopy

The rheology of neutrophils in their passive and activated states plays a key role in determining their function in response to inflammatory stimuli. Atomic force microscopy was used to study neutrophil rheology by measuring the complex shear modulus G*(omega) of passive nonadhered rat neutrophils on poly(HEMA) and neutrophils activated through adhesion to glass. G*(omega) was measured over three frequency decades (0.1-102.4 Hz) by indenting the cells 500 nm with a spherical tip and then applying a 50-nm amplitude multi-frequency signal. G*(omega) of both passive and adhered neutrophils increased as a power law with frequency, with a coupling between elastic (G') and loss (G') moduli. For passive neutrophils at 1.6 Hz, G' = 380 +/- 121 Pa, whereas G' was fourfold smaller and the power law coefficient was of x = 1.184. Adhered neutrophils were over twofold stiffer with a lower slope (x = 1.148). This behavior was adequately described by the power law structural damping model but not by liquid droplet and Kelvin models. The increase in stiffness with frequency may modulate neutrophil transit, arrest, and transmigration in vascular microcirculation.