Multi-Electrode Array with a Planar Surface for Cell Patterning by Microprinting.

Multielectrode arrays (MEAs) are devices for non-invasive electrophysiological measurements of cell populations. This paper describes a novel fabrication method of MEAs with a fully planar surface. The surface of the insulation layer and the surface of the electrodes were on one plane; we named this device the planar MEA (pMEA). The main advantage of the pMEA is that it allows uniform contact between the pMEA surface and a substrate for positioning of microfluidic channels or microprinting of a cell adhesive layer. The fabrication of the pMEA is based on a low adhesive Au sacrificial peel-off layer. In divergence from conventional MEAs with recessed electrodes, the electrodes of the pMEA lead across the sloped edge of the insulation layer. To make this, the profile of the edge of the insulation layer was measured and the impedance of the planar electrodes was characterized. The impedance of the pMEA was comparable with the impedance of conventional MEA electrodes. The pMEA was tested for patterning HL-1 cells with a combination of imprinting fibronectin and coating by antifouling poly (l-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG). The HL-1 cells remained patterned even at full confluency and presented spontaneous and synchronous beating activity.

The Effect of Rhodamine-Derived Superparamagnetic Maghemite Nanoparticles on the Motility of Human Mesenchymal Stem Cells and Mouse Embryonic Fibroblast Cells.

Nanoparticles have become popular in life sciences in the last few years. They have been produced in many variants and have recently been used in both biological experiments and in clinical applications. Due to concerns over nanomaterial risks, there has been a dramatic increase in investigations focused on safety research. The aim of this paper is to present the advanced testing of rhodamine-derived superparamagnetic maghemite nanoparticles (SAMN-R), which are used for their nontoxicity, biocompatibility, biodegradability, and magnetic properties. Recent results were expanded upon from the basic cytotoxic tests to evaluate cell proliferation and migration potential. Two cell types were used for the cell proliferation and tracking study: mouse embryonic fibroblast cells (3T3) and human mesenchymal stem cells (hMSCs). Advanced microscopic methods allowed for the precise quantification of the function of both cell types. This study has demonstrated that a dose of nanoparticles lower than 20 µg·cm-2 per area of the dish does not negatively affect the cells' morphology, migration, cytoskeletal function, proliferation, potential for wound healing, and single-cell migration in comparison to standard CellTracker™ Green CMFDA (5-chloromethylfluorescein diacetate). A higher dose of nanoparticles could be a potential risk for cytoskeletal folding and detachment of the cells from the solid extracellular matrix.

Rhodamine bound maghemite as a long-term dual imaging nanoprobe of adipose tissue-derived mesenchymal stromal cells.

In the last few years, magnetically labeled cells have been intensively explored, and non-invasive cell tracking and magnetic manipulation methods have been tested in preclinical studies focused on cell transplantation. For clinical applications, it is desirable to know the intracellular pathway of nanoparticles, which can predict their biocompatibility with cells and the long-term imaging properties of labeled cells. Here, we quantified labeling efficiency, localization, and fluorescence properties of Rhodamine derivatized superparamagnetic maghemite nanoparticles (SAMN-R) in mesenchymal stromal cells (MSC). We investigated the stability of SAMN-R in the intracellular space during a long culture (20 days). Analyses were based on advanced confocal microscopy accompanied by atomic absorption spectroscopy (AAS) and magnetic resonance imaging. SAMN-R displayed excellent cellular uptake (24 h of labeling), and no toxicity of SAMN-R labeling was found. 83% of SAMN-R nanoparticles were localized in lysosomes, only 4.8% were found in mitochondria, and no particles were localized in the nucleus. On the basis of the MSC fluorescence measurement every 6 days, we also quantified the continual decrease of SAMN-R fluorescence in the average single MSC during 18 days. An additional set of analyses showed that the intracellular SAMN-R signal decrease was minimally caused by fluorophore degradation or nanoparticles extraction from the cells, main reason is a cell division. The fluorescence of SAMN-R nanoparticles within the cells was detectable minimally for 20 days. These observations indicate that SAMN-R nanoparticles have a potential for application in transplantation medicine.

WITHDRAWN: Autologous adipose tissue-derived stromal vascular fraction cells application in patients with osteoarthritis.

Ahead of Print article withdrawn by publisher

Mesenchymal stromal cell labeling by new uncoated superparamagnetic maghemite nanoparticles in comparison with commercial Resovist--an initial in vitro study.

OBJECTIVE: Cell therapies have emerged as a promising approach in medicine. The basis of each therapy is the injection of 1-100×10(6) cells with regenerative potential into some part of the body. Mesenchymal stromal cells (MSCs) are the most used cell type in the cell therapy nowadays, but no gold standard for the labeling of the MSCs for magnetic resonance imaging (MRI) is available yet. This work evaluates our newly synthesized uncoated superparamagnetic maghemite nanoparticles (surface-active maghemite nanoparticles - SAMNs) as an MRI contrast intracellular probe usable in a clinical 1.5 T MRI system. METHODS: MSCs from rat and human donors were isolated, and then incubated at different concentrations (10-200 µg/mL) of SAMN maghemite nanoparticles for 48 hours. Viability, proliferation, and nanoparticle uptake efficiency were tested (using fluorescence microscopy, xCELLigence analysis, atomic absorption spectroscopy, and advanced microscopy techniques). Migration capacity, cluster of differentiation markers, effect of nanoparticles on long-term viability, contrast properties in MRI, and cocultivation of labeled cells with myocytes were also studied. RESULTS: SAMNs do not affect MSC viability if the concentration does not exceed 100 µg ferumoxide/mL, and this concentration does not alter their cell phenotype and long-term proliferation profile. After 48 hours of incubation, MSCs labeled with SAMNs show more than double the amount of iron per cell compared to Resovist-labeled cells, which correlates well with the better contrast properties of the SAMN cell sample in T2-weighted MRI. SAMN-labeled MSCs display strong adherence and excellent elasticity in a beating myocyte culture for a minimum of 7 days. CONCLUSION: Detailed in vitro tests and phantom tests on ex vivo tissue show that the new SAMNs are efficient MRI contrast agent probes with exclusive intracellular uptake and high biological safety.

Flexibility of human cytochrome P450 enzymes: molecular dynamics and spectroscopy reveal important function-related variations.

To gain more complete insight into flexibility and malleability of five forms of human liver cytochrome P450 enzymes, which play major roles in drug metabolism (CYPs 1A2, 2A6, 2C9, 2D6 and 3A4), we employed UV/VIS and resonance Raman spectroscopy in combination with all-atomic molecular dynamics simulations under normal and high pressure conditions (300 MPa). In general, the high pressure reduces the flexibility of CYPs, which become more dense and compact as their radii of gyration and temperature B-factors diminish. The flexibility of CYPs spans the regions, which are localized in solvent exposed loops. A considerable degree of flexibility is also observed at amino-acids making the pw2 and solvent channels, which are suggested to serve for substrate access and/or product release. The number of water molecules as well as the number of protein backbone atoms of the active site in close proximity of heme cofactor generally increases under high pressure. This finding provides new insights regarding the interpretation of pressure-related Soret band red shifts. Presented results also point towards considerable differences between the CYP forms studied: CYP2A6 and CYP1A2 have the least malleable active sites while those of CYP2D6, CYP2C9 and CYP3A4 have considerably greater degrees of flexibility or malleability. In addition, the number of water molecules in the active site cavity of CYP3A4 anomalously decreases under high pressure due to opening of the active site. These results correlate with the known substrate promiscuity of the respective CYP forms, with CYP3A4 displaying the highest substrate promiscuity, corresponding to the most open and malleable active site, whereas CYP1A2 and CYP2A6 show a high substrate-specificity and have a small and rigid active sites.

Flexibility of human cytochromes P450: molecular dynamics reveals differences between CYPs 3A4, 2C9, and 2A6, which correlate with their substrate preferences.

Molecular dynamics (MD) simulations at normal and high temperature were used to study the flexibility and malleability of three microsomal cytochromes P450 (CYPs): CYP3A4, CYP2C9, and CYP2A6. Comparison of B-factors (describing the atomic fluctuations) between X-ray and MD data shows that the X-ray B-factors are significantly lower in the regions where the crystal contacts occur than for other regions. Consequently, the conclusions about CYP flexibility based solely on the X-ray data might be misleading. Comparison of flexibility patterns of the three CYPs enabled common features and variations in flexibility and malleability of the studied CYPs to be identified. The previously described pattern of flexibility in topological elements of microsomal CYPs (a rigid heme binding core, a malleable distal side and intermediately flexible proximal side) was confirmed. These topological features provide an important combination of high stereo- and regio-specificity (mediated by the relative rigidity in the neighborhood of the heme), together with high substrate promiscuity due to the more flexible active site and the malleability of the distal side. The data acquired here show that the malleability of the three studied CYPs correlates with their substrate specificity: CYP2A6 has a narrow substrate range and is the most rigid, CYP3A4 is the most promiscuous CYP known and is the most malleable, and CYP2C9 is intermediate in terms of both its substrate specificity and malleability. Thus, the malleability of CYPs is probably a major determinant of their substrate specificity.

What common structural features and variations of mammalian P450s are known to date?

Sufficient structural information on mammalian cytochromes P450 has now been published (including seventeen X-ray structures of these enzymes by June 2006) to allow characteristic features of these enzymes to be identified, including: (i) the presence of a common fold, typical of all P450s, (ii) similarities in the positioning of the heme cofactor, (iii) the spatial arrangement of certain structural elements, and (iv) the access/egress paths for substrates and products, (v) probably common orientation in the membrane, (vi) characteristic properties of the active sites with networks of water molecules, (vii) mode of interaction with redox partners and (viii) a certain degree of flexibility of the structure and active site determining the ease with which the enzyme may bind the substrates. As well as facilitating the identification of common features, comparison of the available structures allows differences among the structures to be identified, including variations in: (i) preferred access/egress paths to/from the active site, (ii) the active site volume and (iii) flexible regions. The availability of crystal structures provides opportunities for molecular dynamic simulations, providing data that are apparently complementary to experimental findings but also allow the dynamic behavior of access/egress paths and other dynamic features of the enzymes to be explored.