Application of the micropipette technique to the measurement of cultured porcine aortic endothelial cell viscoelastic properties

The viscoelastic deformation of porcine aortic endothelial cells grown under static culture conditions was measured using the micropipette technique. Experiments were conducted both for control cells (mechanically or trypsin detached from the substrate) and for cells in which cytoskeletal elements were disrupted by cytochalasin B or colchicine. The time course of the aspirated length into the pipette was measured after applying a stepwise increase in aspiration pressure. To analyze the data, a standard linear viscoelastic half-space model of the endothelial cell was used. The aspirated length was expressed as an exponential function of time. The actin microfilaments were found to be the major cytoskeletal component determining the viscoelastic response of endothelial cells grown in static culture.     

Application of the microarray technique to cell blocks

OBJECTIVE: To evaluate whether the technique ofmicroarray construction can be applied to paraffin-embedded cell block specinmens. STUDY DESIGN: We used a manual microarray method for construction of a well-aligned cell microarray. First we evaluated the cellularity of the cell block and assigned the cases to the null, low, moderate or high cellularity category. Second, based on the different cellularity, we constructed 25 specimen cylinders into 1 block. We used routine hematoxylin-eosin (H-E) stain and immunocytochemistry (ICC) on the slide. RESULTS: All cell microarray paraffin block shaping and specimen cylinder arraying were finished in 1 step, so the specimen cylinders and the paraffin blocks of the cell microarray could very easily be incorporated. No sample was lost during the H-E staining process. However, a few samples fell off partially during the ICC process. Additionally, we observed that the higher cellularity groups yielded a better outcome as compared to the lower cellularity groups. CONCLUSION: This study introduced a very simple and economical technique for the creation of cell microarrays from cell blocks. This procedure should acquaint cytopathologists with microarray technology and encourage its use.     

T cell locomotion in the tumor microenvironment. I. A collagen-matrix assay

Previous studies have shown that lymphocytic infiltrates of different mouse mammary tumors contain different proportions of the T cell subsets Lyt-1+ and Lyt-2+. These characteristic subset ratios may be established, at least in part, by differential locomotion of subsets in response to components of the tumor microenvironment, such as soluble chemotactic and chemokinetic factors, cell and stromal surfaces, oxygen tension, and pH. We describe a new in vitro assay for determining how such microenvironmental variables affect lymphocyte locomotion. Suspended lymph node cells, alone or mixed with other cell types, are sandwiched between two layers of type I collagen gel and bathed in culture medium. A halo of locomotory cells fans out around the flattened droplet. Locomotion requires energy and exogenous protein. After a 24-hr incubation at 37 degrees C, 10% CO2 in air, the cell density of the halo is analyzed optically and the subset ratios are characterized by immunofluorescence staining of the gel sandwich. Under standard culture conditions, the locomotory population is enriched 12% in Thy-1+ cells compared with the bulk population, and the ratio of Lyt-1+ to Lyt-2+ cells is significantly increased. Lymphocyte locomotion is inhibited by 1 microM PGE2, by decreased pH and oxygen tension, and by the presence of normal mammary cells or mammary adenocarcinoma cells. The Lyt-1+:2+ ratio in the locomotory population is not altered by PGE2 but is reduced by acidity and hypoxia. The ratio is also reduced by the presence of mammary cells and cells of one of the mammary adenocarcinoma cell lines tested (168) but not by two others (68H and 410). Our data support the hypothesis that the locomotion of T cell subsets is differentially responsive to the types of microenvironmental conditions that vary among tumors.     

Analysis of cell locomotion

The methods of statistical physics have been applied to the analysis of cell movement. Human polymorphonuclear leukocytes were exposed to different surfaces possessing parallel oriented physical stuctures (scratched glass surface, machine drilled aluminium surface, optical grid and stretched polyethylene foil) and cell migration was observed using time-lapse photography.We demonstrate that in cell migration along physical structures, referred to as contact guidance, two subgroups can be distinguished: 1) The nematic type where the cell size is large in relation to the grid distance of the undulate surface. 2) The smectic type where the cell size is small in relation to the grid distance of the substrate.Nematic contact guidance is characterized by an anisotropic random walk. In all substrates investigated the diffusion process parallel to the lines was faster than the diffusion process perpendicular to them. The angular dependent diffusion coefficient was described by an ellipse. Deviation from a circle defined an apolar order parameter, whose value was about 0.3. The amount of information which the cells collected from, the undulate surface was very low, between 0.1 and 0.2 bits. We demonstrate that cells do not recognize all the details of their surroundings and that their migration can be compared to the groping around of a short sighted man. The blurred environment can be described by a mean field whose strength is proportional to the aploar order parameter. It is argued that the anisotropic surface tension is the basic source for nematic contact guidance.Smectic contact guidance is characterized by an anisotropic random walk and is quantified by a density order paramter which is 0.28 in the case of the scratched glass surface of a Neubauer counting chamber. The information which the cells collect from their environment is very low (0.03 bits). The lines seen by the cell can be described by a mean field whose strength is proportional to the density oder parameter.Finally, we demonstrate that the locomotion of granulocytes is governed by an internal clock and internal programs. After migrating for a certain time (32 s) in a particular direction, a new direction of locomotion is determined by an internal program. The cell decides basically between left or right, thereby preferring a turn angle such that the cell migrates either parallel or perpendicular to the lines. the angles are nearly equally probable but the cell moves, in the case of nematic guidance, with different velocities in the +or-direction. The cell also has directional memories with characteristic times of 32 s and greater than 100 s.     

Latex bread technique for detection of the plaque-forming cell response to teratocarcinoma tumor antigens

A latex bead technique modified for measuring the plaque-forming cell (PFC) response to teratocarcinoma tumor antigens in syngeneic animals is described. With this method one can detect both the primary (IgM) and the secondary (IgG) immune response to tumor antigens. Optimal detection of the PFC response depends on the proper ratio of sheep red blood cells to latex beads and the dose of tumor cell antigen used for immunization. The presence of fetal calf serum interfered with immunization of animals and the coating of the latex beads with the tumor cell antigens. The plaques obtained in response to immunization with teratocarcinoma cell antigens varied in size, probably reflecting the complex immune response to more than one class of antigens on tumor cells.     

Latex bread technique for detection of the plaque-forming cell response to teratocarcinoma tumor antigens

Summary A latex bead technique modified for measuring the plaque-forming cell (PFC) response to teratocarcinoma tumor antigens in syngeneic animals is described.With this method one can detect both the primary (IgM) and the secondary (IgG) immune response to tumor antigens. Optimal detection of the PFC response depends on the proper ratio of sheep red blood cells to latex beads and the dose of tumor cell antigen used for immunization. The presence of fetal calf serum interfered with immunization of animals and the coating of the latex beads with the tumor cell antigens. The plaques obtained in response to immunization with teratocarcinoma cell antigens varied in size, probably reflecting the complex immune response to more than one class of antigens on tumor cells.     

Application of irreversible thermodynamics to a functional description of the tumor cell membrane

The permeability of the membranes of several strains of Ehrlich mouse ascites tumor cells to a series of homologous glycols was analyzed with equations from irreversible thermodynamics. Experimental data were fitted to the appropriate equations of Kedem and Katchalsky by means of an analog computer and values for σ, L_p, and ωRT were calculated. These parameters would become part of a functional profile of the membrane of the tumor cell. The glycols cross the membrane with energies of activation ranging from 16 kcal/mole for ethylene glycol to 20 kcal/mole for triethylene glycol. Entropies of activation ranged from 17 to 27 entropy units. This analysis provided several additional dividends. It is one of the few examples with a mammalian cell where the kinetics of non-electrolyte transport have been described completely by the equations of irreversible thermodynamics. It has provided the means to distinguish how much of the transport occurred through porous paths in the membrane and how much involved solution within the matrix of the membrane. Finally, several tumor cell populations have been distinguished through these functional properties of their membranes.     

Application of the immunoperoxidase technique to cell block preparations from fine needle aspirates

Immunocytochemistry on fine needle aspiration (FNA) material has been mainly performed on cytologic preparations; there have been few reports on the use of FNA cell blocks. This study compared the intensity scores of immunoperoxidase staining on FNA cell block preparations from 21 breast, 12 thyroid and 10 lymph node aspirates with the scores on the corresponding surgically excised specimens. FNA materials for cell blocks were fixed in formalin and embedded in agar. Ten commercially available antibodies forming three panels were studied using standard peroxidase-antiperoxidase and avidin-biotin complex techniques. In general, the staining results on the FNA cell block sections agreed with those on the surgical specimens; in addition, there were fewer aberrant positive staining results and much less background staining in the cell block sections. These phenomena were most striking with the cytokeratin antibodies. It is concluded that immunoperoxidase staining on FNA cell block preparations is reliable; the advantages of the use of cell block sections as opposed to smears are discussed.     

Direct evidence that cancer cell locomotion contributes importantly to invasion

This study was undertaken to clarify whether active locomotion of cancer cells is important for their ability to invade. The most rapidly moving cells were isolated from a cultured murine parent fibrosarcoma by successive cycles of migration through a micropore membrane. Cells were isolated by unstimulated locomotion and by haptotaxis to laminin, and the selected cells did indeed constitute rapidly locomoting subpopulations. These cells invaded biological tissues more efficiently than did the unselected parent cells. The cells selected by haptotaxis to laminin invaded most rapidly through amnion with basement membranes (containing laminin). Cancer cell haptotaxis to laminin in basement membranes thus promotes penetration of these tissue barriers. These results show in a direct manner that cancer cell locomotion is in fact important in invasion of biological tissues.     

Analysis of cell locomotion on ligand gradient substrates

Directional cell motility plays a key role in many biological processes like morphogenesis, inflammation, wound repair, angiogenesis, immune response, and tumor metastasis. Cells respond to the gradient in surface ligand density by directed locomotion towards the direction of higher ligand density. Theoretical models which address the physical basis underlying the regulatory effect of ligand gradient on cell motility are highly desirable. Predictive models not only contribute to a better understanding of biological processes, but they also provide a quantitative interconnection between cell motility and biophysical properties of the extracellular matrix (ECM) for rational design of biomaterials as scaffolds in tissue engineering. In this work, we consider a one‐dimensional (1D) continuum viscoelastic model to predict the cell velocity in response to linearly increasing density of surface ligands on a substrate. The cell is considered as a 1D linear viscoelastic object with position dependent elasticity due to the variation in actin network density. The cell–substrate interaction is characterized by a frictional force, controlled by the density of ligand–receptor pairs. The generation of contractile stresses is described in terms of kinetic equations for the reactions between actins, myosins, and guanine nucleotide regulatory proteins. The model predictions show a reasonable agreement with experimentally measured cell speeds, considering biologically relevant values for the model parameters. The model predicts a biphasic relationship between cell speed and slope of gradient as well as a maximum limiting speed after a finite migration time. For a given slope of ligand gradient, the onset of the limiting speed appears at longer times for substrates with lower ligand gradients. The model can be applied to the design of biomaterials as scaffolds for guided tissue regeneration as it predicts an optimum range for the slope of ligand gradient. Biotechnol. Bioeng. 2009;103: 424–429. © 2009 Wiley Periodicals, Inc.     

Ruffling and locomotion: Role in cell resistance to growth factor-induced proliferation

It has long been known that the growth rate of cells in vitro can be retarded by providing substrates of restricted area. Such experiments were performed with adhesive islets, made by depositing metals onto agarose layers through templates of various sizes. Since normal cells are unable to adhere to agarose, they become confined to the metallic surface. Using such haptotactic islets, we have studied the role of membrane ruffling and cell locomotion in the resistance of AG1523 human fibroblasts to growth factor-induced mitogenesis. Cells plated on small substrates, i.e., 2,150 μm² in area, initially showed vigorous ruffling, which was suppressed by 8 h after plating but had resumed again by 12 h. In contrast, cells on larger-size islets showed a rapid decline and stabilization of ruffling activity. When the growth rate was measured for single cells cultured on haptotactic islets, it was found to increase linearly from areas of 4,280 μm² up to 425,000 μm². Since the area needed to saturate the growth response was ～50-fold larger than the area occupied by a single cell, the growth inhibition was attributed in part to an interference with locomotion. The implication that locomotion provided positive input into growth control mechanisms was subjected to a direct test by evaluating the effect of nine polypeptide growth factors on the motility of serum-starved cells. All except TGF-β₁ stimulated movement. Finally, the mitogenic effect of growth factors was measured by [³H]thymidine incorporation and found to be proportional to motile activities, as quantitatively assayed. We conclude that locomotion suppression is a factor in AG1523 cell resistance to growth factor-induced mitogenesis. © 1993 Wiley-Liss, Inc.     

From neuromuscular activation to end-point locomotion: An artificial neural network-based technique for neural prostheses

Neuroprostheses, implantable or non-invasive ones, are promising techniques to enable paralyzed individuals with conditions, such as spinal cord injury or spina bifida (SB), to control their limbs voluntarily. Direct cortical control of invasive neuroprosthetic devices and robotic arms have recently become feasible for primates. However, little is known about designing non-invasive, closed-loop neuromuscular control strategies for neural prostheses. Our goal was to investigate if an artificial neural network-based (ANN-based) model for closed-loop-controlled neural prostheses could use neuromuscular activation recorded from individuals with impaired spinal cord to predict their end-point gait parameters (such as stride length and step width). We recruited 12 persons with SB (5 females and 7 males) and collected their neuromuscular activation and end-point gait parameters during overground walking. Our results show that the proposed ANN-based technique can achieve a highly accurate prediction (e.g., R-values of 0.92–0.97, ANN (tansig+tansig) for single composition of data sets) for altered end-point locomotion. Compared to traditional robust regression, this technique can provide up to 80% more accurate prediction. Our results suggest that more precise control of complex neural prostheses during locomotion can be achieved by engaging neuromuscular activity as intrinsic feedback to generate end-point leg movement. This ANN-based model allows a seamless incorporation of neuromuscular activity, detected from paralyzed individuals, to adaptively predict their altered gait patterns, which can be employed to provide closed-loop feedback information for neural prostheses.