Neural stem cells from human cord blood on bioengineered surfaces--novel approach to multiparameter bio-tests.

Stem cell technology combined with emerging surface nano/micro-technologies provides a new tool for better understanding of the mechanisms involved in cell fate decisions and compound-induced adverse reactions. This article provides state-of-the-art on the development of modern multiparameter bio-tests based on interactions between neural stem cells derived from human cord blood and bioengineered surfaces. Cell growth platforms with controlled content, geometry and spatial distribution of bioactive and stem cell attractive areas were fabricated either by micro-contact printing or piezoelectric spotting of polycationic biomolecules or extracellular matrix proteins (ECM) on cell-repellent surfaces. HUCB-NSCs were shown to adhere, differentiate and respond to neurotoxic MeHgCl on functional domains in a manner dependent on protein type and concentration, cell density and serum conditions. While receptor-mediated interactions with ECM proteins under absence of serum promote neuronal differentiation, non-specific adhesion to polycationic molecules maintain cells attached to the surface in non-differentiated stage. Functional domains were further engineered to create "smart" microenvironment by immobilizing to the surface signaling molecules together with ECM proteins. Stimulation of selected intracellular pathways by molecules of Wnt, Shh, CNTF or Notch type resulted in differentiation of HUCB-NSC to either neuronal or astroglial lineage. Sensor techniques applied to HUCB-NSC included measurements of electrical activity using multielectrode array chips. Spontaneous electrical field potentials of HUCB-NSCs were dependent upon developmental stage of tested cells. Bioengineered surfaces, on protein microarrays and micro-electrode array chips provide a novel approach to the multiparameter bio-tests by adding an important information on the sensitivity of certain molecular pathways and functional cellular responses to selected neurotoxins.

Stem-cell culture on patterned bio-functional surfaces.

Bio-functional surfaces have been created by printing proteins on antifouling surfaces in a customised geometry. Human umbilical cord neural stem cells incubated on the samples readily attach to the protein defined domains, where they have been monitored during 21 days of culture. The stability of the pattern varies with the density of cells anchored to the microstamped proteins. Highly packed cell patterned domains favoured non-differentiated mode, while low-density areas allowed the spreading out of the cells and differentiation. Tailoring the geometry (pattern size and distances) enables improving the monitoring of the stem cells' developmental processes. The biocompatible surfaces can serve as a model to study processes accompanying stem cell neural lineage commitment.

Online monitoring of BALB/3T3 metabolism and adhesion with multiparametric chip-based system.

A multiparametric chip-based system was employed to measure cell adhesion, metabolism, and response to metal compounds previously classified as cytotoxic in immortalized mouse fibroblasts (BALB/3T3 cell line). The system measures in parallel, online, and in label-free conditions the extracellular acidification rates (with pH-sensitive field effect transistors [ISFETs]), the cellular oxygen consumption (with amperometric electrode structures [Clark-type sensors]), and cell adhesion (with impedimetric interdigitated electrode structures [IDESs]). The experimental protocol was optimized to monitor metabolism and adhesion of the BALB/3T3 cell line. A total of 70,000 cells and a bicarbonate buffer-free running low-glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal clone serum III and 1mM Hepes were selected to maintain cells in good conditions on the chip during the measurements performed under perfusion conditions. Cells were exposed to sodium arsenite, cadmium chloride, and cis-platinum at concentrations ranging from 1 to 100 microM. The kinetics of cell response to these compounds was analyzed and suggests that the Clark-type sensors can be more sensitive than IDESs and ISFETs in detecting the presence of high chemical concentration when short exposure times (i.e., 2h) are considered. The cytotoxicity data obtained from the online measurements of acidification, respiration, and adhesion at 24h compare well, in terms of half-inhibition concentration values (IC(50)), with the ones obtained using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test and colony-forming efficiency (CFE) assay. The results show a good sensitivity of the system combined with the advantages of the online and label-free detection methods that allow following cell status before, during, and after the treatment in the same experiment.

Effects of zafirlukast on bronchial asthma and allergic rhinitis.

Asthma and allergic rhinitis are common conditions, occurring with increasing prevalence and frequently coexist. In both conditions histamine and cysteinil leukotrienes are important pathogenic inflammatory mediators. We evaluated the effects of the leukotriene receptor antagonist zafirlukast, 20mg administered twice daily for 2 weeks, in patients with allergic rhinitis and bronchial asthma during the grass pollen season. Patients underwent skin prick testing, spirometry, rhinomanometry, mucus transport test with saccharine, nasal epithelial brushing to study ciliary beat and, finally, nasal lavage.Thirty-five subjects completed the study. At the end of the study period, zafirlukast significantly reduced asthma and rhinitis symptoms (P< or =0.05); FEV(1) values were unchanged (P=0.10), whereas nasal resistances showed a decrease following treatment (P=0.01). Ciliary beat frequency (CBF) also improved (P=0.00), although mucociliary transport showed no improvement (P=0.87). The number of eosinophils in nasal lavage fluid decreased (P=0.00) while that of neutrophils was unchanged (P=0.09). These positive effects suggest that zafirlukast may be usefully employed in the treatment of both bronchial asthma, as previously demonstrated, and allergic rhinitis.

New adsorbed coatings for capillary electrophoresis.

New acrylic polymers bearing oxirane groups were synthesized to be used in the production of coated capillaries. A fully automated coating procedure was devised based on the use of diluted water solutions of these polymers. The whole procedure required less than 30 min. The new polymers rapidly adsorbed from water onto the capillary wall, thus suppressing electroosmotic flow (EOF) to a negligible value. The adsorbed coatings were stable for hundreds of hours at high pH, temperature, and in the presence of 8 M urea. Efficient separations of acidic and basic proteins were achieved in the new phases.