Article
Refine
Year of publication
Document Type
- Article (85) (remove)
Has Fulltext
- no (85) (remove)
Keywords
- CellDrum (3)
- Cardiac myocytes (1)
- Cell permeability (1)
- Cellular force (1)
- Circular Dichroism (1)
- Contractile tension (1)
- Endothelial cells (1)
- Heart tissue culture (1)
- Induced pluripotent stem cells (1)
- Inotropic compounds (1)
- Ion channels (1)
- LPS (1)
- Lipopolysaccharide (1)
- NONOate (1)
- Nitric Oxide (1)
- Nitric Oxide Donor (1)
- Pharmacology (1)
- Recombinant activated protein C (1)
- Septic cardiomyopathy (1)
- actin cytoskeleton (1)
A microscopic photometric method for measuring erythrocyte deformability. Artmann, Gerhard Michael
(1986)
Advances in polymer science have significantly increased polymer applications in life sciences. We report the use of free-standing, ultra-thin polydimethylsiloxane (PDMS) membranes, called CellDrum, as cell culture substrates for an in vitro wound model. Dermal fibroblast monolayers from 28- and 88-year-old donors were cultured on CellDrums. By using stainless steel balls, circular cell-free areas were created in the cell layer (wounding). Sinusoidal strain of 1 Hz, 5% strain, was applied to membranes for 30 min in 4 sessions. The gap circumference and closure rate of un-stretched samples (controls) and stretched samples were monitored over 4 days to investigate the effects of donor age and mechanical strain on wound closure. A significant decrease in gap circumference and an increase in gap closure rate were observed in trained samples from younger donors and control samples from older donors. In contrast, a significant decrease in gap closure rate and an increase in wound circumference were observed in the trained samples from older donors. Through these results, we propose the model of a cell monolayer on stretchable CellDrums as a practical tool for wound healing research. The combination of biomechanical cell loading in conjunction with analyses such as gene/protein expression seems promising beyond the scope published here.
Biocompatibility, flexibility and durability make polydimethylsiloxane (PDMS) membranes top candidates in biomedical applications. CellDrum technology uses large area, <10 µm thin membranes as mechanical stress sensors of thin cell layers. For this to be successful, the properties (thickness, temperature, dust, wrinkles, etc.) must be precisely controlled. The following parameters of membrane fabrication by means of the Floating-on-Water (FoW) method were investigated: (1) PDMS volume, (2) ambient temperature, (3) membrane deflection and (4) membrane mechanical compliance. Significant differences were found between all PDMS volumes and thicknesses tested (p < 0.01). They also differed from the calculated values. At room temperatures between 22 and 26 °C, significant differences in average thickness values were found, as well as a continuous decrease in thicknesses within a 4 °C temperature elevation. No correlation was found between the membrane thickness groups (between 3–4 µm) in terms of deflection and compliance. We successfully present a fabrication method for thin bio-functionalized membranes in conjunction with a four-step quality management system. The results highlight the importance of tight regulation of production parameters through quality control. The use of membranes described here could also become the basis for material testing on thin, viscous layers such as polymers, dyes and adhesives, which goes far beyond biological applications.
The CellDrum technology (The term 'CellDrum technology' includes a couple of slightly different technological setups for measuring lateral mechanical tension in various types of cell monolayers or 3D-tissue constructs) was designed to quantify the contraction rate and mechanical tension of self-exciting cardiac myocytes. Cells were grown either within flexible, circular collagen gels or as monolayer on top of respective 1-mum thin silicone membranes. Membrane and cells were bulged outwards by air pressure. This biaxial strain distribution is rather similar the beating, blood-filled heart. The setup allowed presetting the mechanical residual stress level externally by adjusting the centre deflection, thus, mimicking hypertension in vitro. Tension was measured as oscillating differential pressure change between chamber and environment. A 0.5-mm thick collagen-cardiac myocyte tissue construct induced after 2 days of culturing (initial cell density 2 x 10(4) cells/ml), a mechanical tension of 1.62 +/- 0.17 microN/mm(2). Mechanical load is an important growth regulator in the developing heart, and the orientation and alignment of cardiomyocytes is stress sensitive. Therefore, it was necessary to develop the CellDrum technology with its biaxial stress-strain distribution and defined mechanical boundary conditions. Cells were exposed to strain in two directions, radially and circumferentially, which is similar to biaxial loading in real heart tissues. Thus, from a biomechanical point of view, the system is preferable to previous setups based on uniaxial stretching.
Background
Minor changes in protein structure induced by small organic and inorganic molecules can result in significant metabolic effects. The effects can be even more profound if the molecular players are chemically active and present in the cell in considerable amounts. The aim of our study was to investigate effects of a nitric oxide donor (spermine NONOate), ATP and sodium/potassium environment on the dynamics of thermal unfolding of human hemoglobin (Hb). The effect of these molecules was examined by means of circular dichroism spectrometry (CD) in the temperature range between 25°C and 70°C. The alpha-helical content of buffered hemoglobin samples (0.1 mg/ml) was estimated via ellipticity change measurements at a heating rate of 1°C/min.
Results
Major results were:
1) spermine NONOate persistently decreased the hemoglobin unfolding temperature T u irrespectively of the Na + /K + environment,
2) ATP instead increased the unfolding temperature by 3°C in both sodium-based and potassium-based buffers and
3) mutual effects of ATP and NO were strongly influenced by particular buffer ionic compositions. Moreover, the presence of potassium facilitated a partial unfolding of alpha-helical structures even at room temperature.
Conclusion
The obtained data might shed more light on molecular mechanisms and biophysics involved in the regulation of protein activity by small solutes in the cell.