Figure 1.
Plasma lithography for cell patterning.
(A) Photolithography is used to form a template of the desired multicellular network. (B) PDMS is poured over the photoresist pattern to create an initial plasma shielding model. (C) PDMS mold is transformed onto a Petri Dish (polystyrene). (D) The plasma surface treatment is used to produce cell-sensitive chemical pattern on the area of PDMS mold. (E) Cell seeding. (F) Cell stimulation.
Figure 2.
Schematic of cell stimulation.
(A) Force probe stimulation. The direction of stimulation is from above the cells, moving 45° to the tissue culture Petri dish surface. (B) Needle stimulation. The direction of movement is from the side, parallel to the tissue culture Petri dish surface.
Figure 3.
Schematic representation of the non-linear intracellular calcium reaction dynamics as a loop in calcium concentration space.
UC1 and UC2 are lower and upper thresholds of intracellular calcium concentration, respectively, which determine the value of calcium release/intake rate constant, k.
Figure 4.
Experiment image and normalized intensity of cells in single fine line subjected to single stimulus.
(A) Image of a finite single fine line of cells. Cells are labeled 1 through 9. The stimulating probe is clearly visible on the right of the stimulated cell (cell 4 labeled with a red circle). The time (in sec) at which the normalized fluorescence reaches its maximum positive rate of change is indicated for every cell. The uncertainty for each one of these times is 0.6 s. The origin of time is the time at which fluorescence in the stimulated cell attains its maximum rate of change. (B) and (C) show the normalized intensity of fluorescence of cells 4 through 9 and cells 3 through 1, respectively, as functions of time. The vertical axis is the dimensionless normalized intensity of fluorescence and the horizontal axis is the time in seconds in intervals of 1.2 s between recordings.
Figure 5.
Simulated calcium concentration for cells in a single chain subjected to a single stimulus.
We label the stimulated cell 4 (cell 31 in the chain of 61 cells). We report the response of cells on either side of the stimulated cell as cell 5 through cell 9 and cell 1-through cell 3 to facilitate comparison with experimental results. The real time is obtained by scaling the cell-to-cell propagation time of the simulation to that of the experiment (see text for details).
Table 1.
Value of dimensionless parameters used in the reaction-diffusion model of intercellular and intracellular reaction-diffusion dynamics.
Figure 6.
Experiment image and normalized intensity of cells in single fine line subjected to double stimulus.
(A) Image of a finite single fine line of cells subjected to dual mechano-stimulation. The stimulating probes are visible at the top-left and bottom-right of the image. The response time of cells labeled in red was calculated from the maximum rate of change of the fluorescence intensity with uncertainties 0.6 s. The response time of cells labeled in green was calculated from a running average of the fluorescence intensity with uncertainties 1.2 s. The first response time for cell 4 (1), represents the time of the first sharp rise in fluorescence versus time. The second response time of cell 4 (2), indicates the time when the fluorescence intensity increases a second time. Cells 4 and 5 represent the region where the calcium pulses are anticipated to meet. (B) and (C) present the normalized intensity of fluorescence of individual cells as a function of time.
Figure 7.
Simulated calcium concentration for cells in a single chain subjected to double stimulus.
Cell 1 and cell 8 are the stimulated cells. We report the response of cells between the stimulated cells as cell 2 through cell 7 to facilitate comparison with experimental results. The real time is obtained by scaling the cell-to-cell propagation time of the simulation to that of the experiment (see text for details).
Figure 8.
Experiment image 1 and normalized intensity of cells in “T” structure subjected to a single mechano-stimulation.
(A) Image of a T structure of cells subjected to single mechano-stimulation. The red circle indicates the location of the stimulated cell. Red labels correspond to cells exhibiting strong fluorescence with response time measured from the rate of change of the fluorescence intensity. Green labels correspond to cells exhibiting weak fluorescence and response time derived from running averages of the rate of change of the fluorescence intensity. Black labels are for cells that show very weak (within the noise level) to no fluorescence. (B–E) shows the normalized intensity of fluorescence of branch 1, cluster area, branch 2 and branch 3, respectively, as functions of time. The vertical axis is the dimensionless normalized intensity of fluorescence and the horizontal axis is the time in seconds in intervals of 1.2 s between recordings.
Figure 9.
Experiment image 2 and normalized intensity of cells in “T” structure subjected to a single mechano-stimulation.
(A) Image of a T structure of cells subjected to single mechano-stimulation. See Figure 8 for detail. (B–D) shows the normalized intensity of fluorescence of representative cells as functions of time. The vertical axis is the dimensionless normalized intensity of fluorescence and the horizontal axis is the time in seconds in intervals of 1.2 s between recordings.
Figure 10.
Schematic illustration and simulation results of the model “T” structure subjected to single stimulus with .
(A) Schematic illustration of the model “T” structure subjected to a single stimulus. Red cell is the stimulated cell. Orange arrows represent the edge-to-edge diffusion. Green arrows represent vertex-to-vertex diffusion. Purple cells highlight the junction cell cluster. (B) Calcium concentration of cells in backbone as a function of time. (C) Calcium concentration of cells 1', 2' and cells in the side branch.
Figure 11.
Simulated calcium concentration for cells in the “T” structure subjected to single stimulation with .
(A) reports the response of cells 28 to 31 in backbone as a function of time. (B) shows the calcium concentration of cells 30 to 33 in backbone as a function of time. Notice the change of scale of the vertical axis. (C) illustrates the calcium concentration of cells 31, 1' and 2' in the junction area and the side branch as a function of time. Again notice the change of scale of the concentration axis.
Figure 12.
Experiment image and normalized intensity of cells in “T” structure subjected to double stimulation.
(A) Image of a T structure of cells subjected to double mechano-stimulation. The red circle identifies the stimulated cells. Red labels indicate the response time individual cells. All the cells exhibited strong fluorescence with response times calculated from the rate of change of the fluorescence intensity. Black labels show cells exhibiting only noise level fluorescence. (B–E) show the normalized intensity of fluorescence of branch 1, cluster area, branch 2 and branch 3, respectively, as functions of time. The vertical axis is the dimensionless normalized intensity of fluorescence and the horizontal axis is the time in seconds in intervals of 1.2 s between recordings.
Figure 13.
Schematic illustration and simulation results of the model “T” structure subjected to double stimulus.
(A) Schematic representation of “T” structure of cells subjected to dual stimulation. Red cells are the stimulated cells. See Figure 10 for additional details. (B) Calcium concentration as a function of time for cells in the vicinity of the junction with .