Dielectrophoretic stretching of cells allows for characterization of their mechanical properties

The mechanical behavior of biological cells is mainly determined by the cytoskeleton. Its properties are closely interlinked with many cellular events, including disease-related processes, and, thus, may be exploited as potent biomarkers. We have stretched two types of cells between microelectrodes through the application of dielectrophoretic forces. Small numbers of cells of cancerous origin (MCF-7) and from related noncancerous tissue (MCF-10A) were sufficient to obtain data that allowed us to unambiguously distinguish these cells. The Maxwell tension applied has been estimated to be 56 Pa. A detailed analysis of the cells showed that the differences in the stretching response are due to cell-specific mechanical properties. Through the addition of an actin- and a microtubule-specific toxin to the cells, differences in the microtubular structures of the two cell types have been identified as the major cause for the behavior observed. Our approach shows enormous potential for parallelization and automation. Hence, it should be suitable for achieving throughputs that make it attractive for many biomedical diagnostic purposes.     

The high dispersion of DNA-multiwalled carbon nanotubes and their properties

DNA-wrapped multiwalled carbon nanotubes (MWCNTs) were successfully obtained by a simple sonication treatment method. The obtained materials were characterized in detail by Raman spectroscopy and scanning electron microscopy (SEM). An SEM image showed that MWCNTs were dispersed sufficiently and covered entirely with DNA. This resulted in high aqueous solubility of the products, with a stability of more than several months. The interaction between DNA and MWCNTs was confirmed by Raman measurements and was ascribed to the strong π-π interactions between the backbones of DNA and the surface of carbon nanotubes. The cyclic voltammograms showed that the composite exhibited excellent electrochemical properties. Experimental results also revealed that the high dispersion of DNA-assisted MWCNTs presented a better property compared with pristine MWCNTs. This facile method for obtaining water-soluble MWCNTs has great potential application for both bioscience and biotechnology.     

Bovine endometrial stromal cells display osteogenic properties

The endometrium is central to mammalian fertility. The endometrial stromal cells are very dynamic, growing and differentiating throughout the estrous cycle and pregnancy. In humans, stromal cells appear to have progenitor or stem cell capabilities and the cells can even differentiate into bone. It is not clear whether bovine endometrial stromal cells exhibit a similar phenotypic plasticity. So, the present study tested the hypothesis that bovine endometrial stromal cells could be differentiated along an osteogenic lineage. Pure populations of bovine stromal cells were isolated from the endometrium. The endometrial stromal cell phenotype was confirmed by morphology, prostaglandin secretion, and susceptibility to viral infection. However, cultivation of the cells in standard endometrial cell culture medium lead to a mesenchymal phenotype similar to that of bovine bone marrow cells. Furthermore, the endometrial stromal cells developed signs of osteogenesis, such as alizarin positive nodules. When the stromal cells were cultured in a specific osteogenic medium the cells rapidly developed the characteristics of mineralized bone. In conclusion, the present study has identified that stromal cells from the bovine endometrium show a capability for phenotype plasticity similar to mesenchymal progenitor cells. These observations pave the way for further investigation of the mechanisms of stroma cell differentiation in the bovine reproductive tract.     

Erythrocyte elastic and mechanical properties and mechanism of their change during irradiation

A decrease in erythrocyte deformation, which was maximum at the height of radiation sickness, was registered as early as 1-3 days following 6 Gy irradiation of rats. The aggregation of erythrocytes increased during the latent period of radiation sickness then sharply decreased. The role of the observed changes in radiation sickness then pathogenesis is discussed. It is established that they are associated with the altered fatty acid composition of the lipid phase of erythrocytic membranes, on the one hand, and with the disturbances in its ionic permeability, on the other.     

Phage display systems and their applications

Screening phage display libraries of proteins and peptides has, for almost two decades, proven to be a powerful technology for selecting polypeptides with desired biological and physicochemical properties from huge molecular libraries. The scope of phage display applications continues to expand. Recent applications and technical improvements driving further developments in the field of phage display are discussed.     

Modelling the mechanical properties of single suspension-cultured tomato cells

BACKGROUND AND AIMS: The relationship between composition and structure of plant primary cell walls, and cell mechanical properties is not fully understood, partly because intrinsic properties of walls such as Young's modulus cannot be obtained readily. The aim of this work is to show that Young's modulus of walls of single suspension-cultured tomato cells can be determined by modelling force-deformation data. METHODS: The model simulates the compression of a cell between two flat surfaces, with the cell treated as a liquid-filled sphere with thin compressible walls. The cell wall and membrane were taken to be permeable, but the compression was so fast that water loss could be neglected in the simulations. Force-deformation data were obtained by compressing the cells in micromanipulation experiments. RESULTS:Good fits were obtained between the model and low-strain experimental data, using the modulus and initial inflation of the cell as adjustable parameters. The mean Young's modulus for 2-week-old cells was found to be 2.3 +/- 0.2 GPa at pH 5. This corresponds to an instantaneous bulk modulus of elasticity of approx. 7 MPa, similar to a value found by the pressure probe method. However, Young's modulus is a better parameter, as it should depend only on the composition and structure of the cell wall, not on bulk cell behaviour. This new method has been used to show that Young's modulus of cultured tomato cell walls is at its lowest at pH 4.5, the pH optimum for expansin activity. CONCLUSIONS:The linear elastic model is very suitable for estimating wall Young's modulus from micromanipulation experiments on single tomato cells. This is a powerful method for determining cell wall material properties.     

Hyperoxia alters the mechanical properties of alveolar epithelial cells

Patients with severe acute lung injury are frequently administered high concentrations of oxygen (>50%) during mechanical ventilation. Long-term exposure to high levels of oxygen can cause lung injury in the absence of mechanical ventilation, but the combination of the two accelerates and increases injury. Hyperoxia causes injury to cells through the generation of excessive reactive oxygen species. However, the precise mechanisms that lead to epithelial injury and the reasons for increased injury caused by mechanical ventilation are not well understood. We hypothesized that alveolar epithelial cells (AECs) may be more susceptible to injury caused by mechanical ventilation if hyperoxia alters the mechanical properties of the cells causing them to resist deformation. To test this hypothesis, we used atomic force microscopy in the indentation mode to measure the mechanical properties of cultured AECs. Exposure of AECs to hyperoxia for 24 to 48 h caused a significant increase in the elastic modulus (a measure of resistance to deformation) of both primary rat type II AECs and a cell line of mouse AECs (MLE-12). Hyperoxia also caused remodeling of both actin and microtubules. The increase in elastic modulus was blocked by treatment with cytochalasin D. Using finite element analysis, we showed that the increase in elastic modulus can lead to increased stress near the cell perimeter in the presence of stretch. We then demonstrated that cyclic stretch of hyperoxia-treated cells caused significant cell detachment. Our results suggest that exposure to hyperoxia causes structural remodeling of AECs that leads to decreased cell deformability.     

Human skin mast cells: their dispersion, purification, and secretory characterization

Digestion of human foreskin with collagenase and hyaluronidase disperses approximately 3.4 X 10(7) nucleated cells per gram of tissue, of which mast cells constitute 4.7%. These may be purified to 80% by use of density gradient centrifugation. The majority of mast cells (79%) measured between 9 and 13 micron in diameter, and the mean histamine content was 4.6 pg/cell. Viability was demonstrated by trypan blue exclusion by 93% of the cells and the low spontaneous histamine secretion of less than 7% in functional studies. Anti-IgE released up to 17.5% of cell-associated histamine within 5 to 7 min. Calcium ionophore-induced release was optimal with 0.3 microM A23187 when 28.6% histamine was released. Unlike human lung mast cells, skin mast cells released histamine in response to compound 48/80 and poly-L-lysine. This release, which was complete within 20 sec, was totally dependent on intact glycolysis and oxidative phosphorylation and partially dependent on extracellular calcium. The same characteristics were observed with secretion induced by substance P and morphine. The weak activity of eledoisin and physalaemin suggests that the substance P receptor, like that of the rat mast cell, is not of the classical types described for smooth muscle. Morphine-induced secretion was partially blocked by naloxone in a manner not compatible with competitive antagonism at a classical opioid receptor. The sensitivity of skin mast cells to nonimmunologic stimulation clearly distinguishes them from mast cells of the lung and lymphoid tissues and provides evidence of functional heterogeneity within human mast cells.     

Physiological salines and the mechanical properties of trout red blood cells

Suspensions of rainbow trout erythrocytes in different physiological salines were compared with respect to their haematological and filtration properties.
A method is described for the suspension of erythrocytes in Cortland saline which has proved suitable for studies of their mechanical properties over periods of several hours.
Significant differences were found between whole blood samples taken during cannulation and after several days recovery, particularly mean cell volume, frequency distribution of red cell volumes and the pore passage time through nucleopore filters. These differences were also found using red cell suspensions of the same bloods. The pore passage time of whole blood sampled during cannulation or its suspensions is less than that of recovery blood although its mean cell volume is greater.     

Force spectroscopy of collagen fibers to investigate their mechanical properties and structural organization

Tendons are composed of collagen and other molecules in a highly organized hierarchical assembly, leading to extraordinary mechanical properties. To probe the cross-links on the lower level of organization, we used a cantilever to pull substructures out of the assembly. Advanced force probe technology, using small cantilevers (length <20 microm), improved the force resolution into the sub-10 pN range. In the force versus extension curves, we found an exponential increase in force and two different periodic rupture events, one with strong bonds (jumps in force of several hundred pN) with a periodicity of 78 nm and one with weak bonds (jumps in force of <7 pN) with a periodicity of 22 nm. We demonstrate a good correlation between the measured mechanical behavior of collagen fibers and their appearance in the micrographs taken with the atomic force microscope.     

Micromanipulation measurement of the mechanical properties of baker's yeast cells

The mechanical properties of a sample of baker's yeast cells were measured by micromanipulation. The relationship between the force required to burst a single cell and its corresponding diameter was established. For stationary phase cells, the compressive force required to burst a cell varied between 55 and 175N, with a mean value of 101 ± 2N. This is a substantial force compared to that required to burst a single mammalian cell (1.5–4.5N), which presumably reflects the lack of a cell wall of the latter. From measurements on 120 cells, there was no significant dependence of bursting force on yeast cell size. The micromanipulation method will be valuable for studying the dependence of mechanical properties of yeast cells on fermentation conditions, and the consequential effects of their behaviour in process disruption operations. © Rapid Science Ltd. 1998     

Dynamic mechanical properties of body-wall dermis in various mechanical states and their implications for the behavior of sea cucumbers

The dermis of the sea cucumber body wall is a typical catch connective tissue that rapidly changes its mechanical properties in response to various stimuli. Dynamic mechanical properties were measured in stiff, standard, and soft states of the sea cucumber Actinopyga mauritiana. Sinusoidal deformations were applied, either at a constant frequency of 0.1 Hz with varying maximum strain of 2%-20% or at a fixed maximum strain of 1.8% with varying frequency of 0.0005-50 Hz. The dermis showed viscoelasticity with both strain and strain-rate dependence. The dermis in the standard state showed a J-shaped stress-strain curve with a stiffness of 1 MPa and a dissipation ratio of 60%; the curve of the stiff dermis was linear with high stiffness (3 MPa) and a low dissipation ratio (30%). Soft dermis showed a J-shaped curve with low stiffness (0.3 MPa) and a high dissipation ratio (80%). The strain-induced softening was observed in the soft state. Stiff samples had a higher storage modulus and a lower tangent delta than soft ones, implying a larger contribution of the elastic component in the stiff state. A simple molecular model was proposed that accounted for the mechanical behavior of the dermis. The model suggested that stiffening stimulation increased inter-molecular bonds, whereas softening stimulation affected intra-molecular bonds. The adaptive significance of each mechanical state in the behavior of sea cucumbers is discussed.     

Determining the mechanical properties of hazel forks by testing their component parts

The ability to accurately predict the load-bearing capacity of tree forks would improve tree surveying and tree surgery techniques and assist with the biomechanical modelling of a tree’s structure. In this study, the bending strength of forks of hazel (Corylus avellana L.) was investigated by assessing the mechanical contributions from three component parts of each fork. Intact forks and ones in which either central or peripheral xylem lying under the branch bark ridge at the apex of the forks had been removed were subjected to tensile tests. The bending strength of these forks was compared with that of the arising branches by carrying out a three-point bending test on the smaller arising branches of the intact specimens. All forks failed in tension, splitting between the arising branches. By removing the centrally placed xylem, constituting approximately a fifth of the width of the fracture surface, the forks’ bending strength was reduced by around 32 %, while removing the outer four-fifths reduced the forks’ bending strength by 49 %. Intact forks had around 74 % of the maximum bending strength of the smaller arising branch. It is concluded that the tensile strength of the centrally placed xylem at the apex of a tree fork is a critical strengthening component. This helps to explain the weakness of forks with included bark, which lack this component. This study concludes that tree forks should not by default be considered flaws in a tree’s structure.     

Atomic force microscopy imaging and mechanical properties measurement of red blood cells and aggressive cancer cells

Mechanical properties play an important role in regulating cellular activities and are critical for unlocking the mysteries of life. Atomic force microscopy (AFM) enables researchers to measure mechanical properties of single living cells under physiological conditions. Here, AFM was used to investigate the topography and mechanical properties of red blood cells (RBCs) and three types of aggressive cancer cells (Burkitt??s lymphoma Raji, cutaneous lymphoma Hut, and chronic myeloid leukemia K562). The surface topography of the RBCs and the three cancer cells was mapped with a conventional AFM probe, while mechanical properties were investigated with a micro-sphere glued onto a tip-less cantilever. The diameters of RBCs are significantly smaller than those of the cancer cells, and mechanical measurements indicated that Young??s modulus of RBCs is smaller than those of the cancer cells. Aggressive cancer cells have a lower Young??s modulus than that of indolent cancer cells, which may improve our understanding of metastasis.     

Mitochondrial dynamics in chondrocytes and their connection to the mechanical properties of the cytoplasm

BACKGROUND: The motion and redistribution of intracellular organelles is a fundamental process in cells. Organelle motion is a complex phenomenon that depends on a large number of variables including the shape of the organelle, the type of motors with which the organelles are associated, and the mechanical properties of the cytoplasm. This paper presents a study that characterizes the diffusive motion of mitochondria in chondrocytes seeded in agarose constructs and what this implies about the mechanical properties of the cytoplasm. METHOD OF APPROACH: Images showing mitochondrial motion in individual cells at 30 s intervals for 15 min were captured with a confocal microscope. Digital image correlation was used to quantify the motion of the mitochondria, and the mean square displacement (MSD) was calculated. Statistical tools for testing whether the characteristic motion of mitochondria varied throughout the cell were developed. Calculations based on statistical mechanics were used to establish connections between the measured MSDs and the mechanical nature of the cytoplasm. RESULTS: The average MSD of the mitochondria varied with time according to a power law with the power term greater than 1, indicating that mitochondrial motion can be viewed as a combination of diffusion and directional motion. Statistical analysis revealed that the motion of the mitochondria was not uniform throughout the cell, and that the diffusion coefficient may vary by over 50%, indicating intracellular heterogeneity. High correlations were found between movements of mitochondria when they were less than 2 microm apart. The correlation is probably due to viscoelastic properties of the cytoplasm. Theoretical analysis based on statistical mechanics suggests that directed diffusion can only occur in a material that behaves like a fluid on large time scales. CONCLUSIONS: The study shows that mitochondria in different regions of the cell experience different characteristic motions. This suggests that the cytoplasm is a heterogeneous viscoelastic material. The study provides new insight into the motion of mitochondria in chondrocytes and its connection with the mechanical properties of the cytoplasm.     

Changes in nano‐mechanical properties of human epidermal cornified cells depending on their proximity to the skin surface

During formation of the stratum corneum (SC) barrier, terminally differentiated keratinocytes continue their maturation process within the dead superficial epidermal layer. Morphological studies of isolated human corneocytes have revealed differences between cornified envelopes purified from the deep and superficial SC. We used atomic force microscopy to measure the mechanical properties of native human corneocytes harvested by tape‐stripping from different SC depths. Various conditions of data acquisition have been tested and optimized, in order to obtain exploitable and reproducible results. Probing at 200 nN allowed us to investigate the total stiffness of the cells (at 50 nm indentation) and that of the cornified envelopes (at 10 to15 nm), and lipid envelopes (at 5 to 10 nm). The obtained data indicated statistically significant differences between the superficial (more rigid) and deep (softer) corneocytes, thus confirming the existence of depth and maturation‐related morphological changes within the SC. The proposed approach can be potentially used for minimally invasive evaluation of various skin conditions such as aging, skin hydration, and pathologies linked to SC.     

Relevance of rheological properties of gel beads for their mechanical stability in bioreactors

The mechanical stability of biocatalyst particles in bioreactors is of crucial importance for applications of immobilized-cell technology in bioconversions. The common methods for evaluation of the strength of polymer beads (mostly force-to-fracture or tensile tests) are, however, not yet proven to be relevant for the assessment of their mechanical stability in bioreactors. Therefore, we tested fracture properties of gel materials and investigated their relevance for abrasion in bioreactors. Abrasion of gel beads was assumed to be a continuous fracturing of the bead surface. At first, three rheological properties were considered: stress at fracture; strain at fracture; and the total fracture energy. If stress at fracture is the most important property, beads having a similar fracture energy, but a smaller stress at fracture, would abrade faster in a bioreactor than beads with a larger stress at fracture; if fracture energy the determining factor, beads that require less energy to fracture would abrade faster than those having a larger fracture energy for the same fracture stress. To determine this, beads of kappa-carrageenan and agar (at two different polymer concentrations) were tested for abrasion in four identical bubble columns under the same operating conditions. Agar beads were expected to abrade faster than those of carrageenan because agar had either a lower stress at fracture or a lower fracture energy. However, no correlation between fracture properties and abrasion rate was found in any of the combinations tested. Carrageenan beads abraded faster than those of agar in all combinations. Furthermore, both the stress and strain at fracture of agar and carrageenan beads decreased during the run and those of carrageenan decreased faster, suggesting that the gels are liable to fatigue in different ways. This hypothesis was confirmed by oscillating experiments in which gel samples were subjected to repeated compressions below their fracture levels. Their resistance to compression clearly decreased with the number of oscillations. Fatigue is probably related to the development of microcracks and microfracture propagation within the material. We concluded that: (a) the use of tests based on bead rupture do not provide relevant information on the mechanical stability of gel beads to abrasion; and (b) abrasion of polymer beads is likely to be related to fatigue of the gel materials. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 517-529, 1997.