illustrates PC-3 cell culture proliferation during the first 72 hours after loading. We took bright-field images of cells cultured inside microfluidic chips using a Nikon TE-2000 inverted microscope. We took pictures every 24 hours in separate chips; shows a representative sequence of pictures from one chip. Each picture covers a 900 micron × 900 micron region. At 0 hours, just after loading, cells floated inside the chamber in round shapes. After incubation for 24 hours, most of the cells had settled and attached to the bottom surface; some of these had begun to spread out. We noticed a small increase in the number of cells. After 48 hours, most cells spread out and attained long spindle shapes. At this point, the number of cells increased significantly. At 72 hours, the cells had grown significantly to near confluence. We counted cells in three different chips to derive cell numbers as a function of time. In , the average number of cells is plotted in blue as a function of culture time. The blue points are the average numbers of cells with error bars representing the corresponding standard errors of means. The increase in cell number fits an exponential growth model (red line), and the total number of cells nearly triples during the 72 hour incubation period. At this time, the chips contained a number of cultured cells adequate for a subsequent passage process.
Fig. 3 (A) Pictures of cell growth inside the culture chamber, where number increases gradually with time (0, 24, 48 and 72 hours). (B) Cell number plotted as a function of time. Blue dots are average cell numbers collected from culture chambers in three different (more ...)
Cancer cell passage is a core function of our chip design. The advantage of our approach is that the chip uses micro-scale shearing forces for detaching adherent cells without the use of chemical reagents, such as trypsin. Hence, this trypsin-free passage no longer consumes the gelatin coating, which enables multiple uses of the growth surface. DMEM supplemented with Fetal Bovine Serum and antibiotics is the only reagent needed, which allows for a simple, robust system. reveals the working mechanism of the diaphragms used for cell passage inside the chip. The left picture is a cross-section view of two adjacent diaphragms in the layered chip structure. There are four electromagnetic (EM) valves, which are divided into two groups controlling the diaphragms individually. The left diaphragm is controlled by the A1 and A2 valves, while the right is controlled by the B1 and B2 valves, both of which share the same gas source. PC-3 cells are cultured on the gelatin-coated glass surface beneath the diaphragms. When A1 is closed and A2 is open, compressed gas will be forced into the left diaphragm chamber causing the pressure to accumulate above the diaphragm, pushing it downward. The volume of the medium decreases as the diaphragm is compressed, creating a hydrodynamic shearing force capable of detaching the cells from the gelatin coated growth surface. Simultaneously in the right diaphragm, the reverse process occurs; B2 is closed and B1 is open. In this case, the compressed air is blocked by B2 and the gas flows externally through B1, decreasing the pressure within the channel, thereby pulling the diaphragm upwards. These diaphragm repelling and imbibing actions are produced continuously via synchronized electrical signals, allowing the volume of the medium inside the chamber to remain constant, as shown in . As the figure shows, we switch every 0.5 seconds between two groups of digital signals operating the four valves, e.g., the direction of forces under each diaphragm changes at a fixed 2 Hz frequency. The upper right inset is the complete setup for the above diaphragm operations. There are six EM valves, four of which are used in this experiment and operated by signals from the lower control box. The gas regulator (Okolab Inc, Ottaviano, Italy) linked to the valves at the side provides constant compressed air with pressure controls.
Fig. 4 (A) Structures and mechanisms of PDMS diaphragms in passaging cancer cells with electromagnetic valves. The photograph shows the control box as well as the gas regulator, which controls the EM valves. (B) Different variations of gas pressures for left (more ...)
A computer recorded diaphragm pressure measured using a manometer (Sper Scientific, Scottsdale, AZ). The manometer connected to the left chamber in . When we applied the first signal (− + + − ) to the valves, the pressure inside the chamber rose. When we applied the second signal (+ − − +), pressure decreased to zero. shows the triangle waves of pressure for maximum pressures of 0.2, 0.4 and 0.6 psi, in green, blue, and red, respectively. The maximum pressure is determined by the gas source. Higher input gas pressure results in higher maximum pressure inside the diaphragm. The triangle waves in remain stable in amplitude and shape during the hundreds of cycles necessary for cell passage. In this way, the diaphragms generate reliable actuated forces for controlled rates of cell detachment during cell passage.
Hydrodynamic forces are the key for detachment of cells during the passage process. shows a numerical simulation that is representative of the fluid motion in the device during passage. Flow was simulated in a numerical solver (COMSOL) with boundary conditions as shown in the central figure. shows the magnitude of the velocity field, where the depressed diaphragm moves downward at 500 microns s−1 while the raised membrane moves upward at the same rate. is a vector-field plot of the 1000 micron region surrounding the lowest point of the diaphragm. The simulation indicates that the fluid moves at greatest speed under the diaphragm and that fluid moves primarily in the horizontal direction, transverse to the diaphragm surface.
Fig. 5 Numerical simulation using COMSOL of fluid motion inside the device during passage. The depressed membrane moves downward at 500 microns s−1 while the raised membrane moves upward at the same rate. (A) Vector-field plot of the 1000 micron region (more ...)
We model cells as 15 micron spheres and estimate hydrodynamic forces 10 microns above the surface of the substrate (white dashed line in the central figure). We compare the relative strengths of two forces: shear force from gradients in the lateral velocity and drag force from the vertical velocity. We neglect drag force from the lateral velocity assuming that confluent cells shield each other from lateral flow. The shear force is estimated by multiplying the shear stress at 10 microns above the surface with the surface area of a cell. The drag force in the vertical direction is similarly estimated with the Stokes’ drag relation using the vertical velocity field. shows a plot of the magnitude of these forces as a function of position in the device. Drag forces are more than an order of magnitude weaker than shear forces. The shear force is greatest under the membrane, dropping from a peak value of 221 pN to a level value of about 50 pN in the open area between diaphragms. This results in relatively fewer cells detached in these open areas. As the diaphragms switch back and forth, cells underneath the diaphragms experience stronger shear forces and are more prone to detachment from the surface.
is one group of microscope images of the PC-3 cell passage process as it occurs inside the cell culture chamber under 0.4 psi and 0.8 psi peak pressures. In our experiments, pictures were taken every second (here those at 0, 10, 40 and 80 seconds are displayed). As can be seen at 0.4 psi immediately after the diaphragms are activated, shearing forces are applied directly to the cells underneath, detaching them from the surface. The number of cells attached to the surface decreases significantly with time, especially during the initial 10 seconds. As time increases, the rate of cell detachment decreases. At 80 seconds, the majority of cells have been detached, leaving the rest as seeds for the next generation of cells. Afterwards, the number of cells remains constant. Passage at 0.8 psi increases the rate of detachment, leaving fewer cells on the surface. At this point, the diaphragms compress the cancer cells with direct contact, flattening them on the surface, as is clearly visible in the picture at 10 seconds. Reintroducing the detached cells into typical cell culture conditions shows that few of these cells are healthy or viable, due to damage resulting from the hard, shearing forces. The blurs in the picture at 80 seconds are traces of floating cells which move rapidly with diaphragm actuations. A lower gas pressure, such as 0.4 psi, is ideal, not only for keeping cells intact under shearing forces, but also for maintaining an adequate number of cells for repeated culture. After detachment, a higher number of cells stayed in the areas underneath the gaps between two neighboring diaphragms than in those areas underneath the diaphragms (data not shown). For example, at 0.4 psi, 60% of cells might remain attached between the diaphragms, compared to 40% beneath the diaphragm. This results from relatively weaker shearing forces between diaphragms, as explained in .
PC-3 cell mechanical passage with different gas pressures. The upper panel shows cells passaged using a peak pressure of 0.4 psi; the lower panel shows rapid cell passage at 0.8 psi.
Additional analysis was done to test the chip’s capacity for repeated trypsin-free passage. We actuated mechanical passage with gas pressures of 0.2 psi, 0.4 psi and 0.8 psi. We performed three separate passages at each gas pressure using the same cell densities and culture conditions. During the passage process, the number of remaining cells imaged by the microscope was counted and the fraction of attached cells was plotted as a function of elapsed time, as shown in . The data points are average numbers of attached cells at specific times, with error bars representing standard errors of means. The fractions decrease with time, as indicated with colored curves to guide the eye. Thus, according to need, percentages and numbers of “seed” cells can be accurately selected by adjusting the duration of the passage with digital controls.
Fractions of attached cells at various times during passage for gas pressures of 0.2, 0.4 and 0.8. psi.
Viability of cells remaining after passage is a key measure of the chip’s ability to be reused for continuous culture. To monitor the ability of the PC-3 cells to regenerate, three chips were passaged under 0.4 psi for 120 seconds and examined afterwards. We counted these cells to derive cell numbers as a function of time. shows a group of cells growing in one location at 0, 24, 48, and 72 hours. In , the number of cells averaged across the three chips is plotted as a function of culture time. Error bars show the corresponding standard errors of means. The number of cells remaining after each passage is greater than the number of cells grown from the initial planting, shown in . Cells eventually reached saturation and could be passaged again.
Fig. 8 (A) Microscope images showing PC-3 cells that remained inside the chip continue to grow after mechanical passaging. The culture surface is fully covered with cells within 48 hours. (B) Cell number plotted as a function of time after passage. Green dots (more ...)
Output cells were also checked for viability. We collected the cells output from the previous passage. We found that these cells were isolated and not clumped. They were mixed with trypan blue (MP biomedicals #1691049) at 1 : 1 ratio. After a five minute incubation, the cells were placed in the hemacytometer and observed under a microscope (). The cells in blue color were dead cells, and cells in white were live. We also counted the cells using an automated cell counter (Contess, Invitrogen, Inc). The result shows that, on average, 97% of cells were live.
Fig. 9 (A) PC-3 cells passaged for the 9th time at 30 days that were collected and tested for viability with trypan blue. The blue cells are dead. The majority of cells are uncolored, indicating they are live. The result shows that the output cells have 97% (more ...)
Long lifetime is another important feature for a culture chip. For this chip, the lifetime is mainly determined by the lifespan of the gelatin coating. To test the longevity of the chips, we used two chips to continuously culture and passage PC-3 cells for 30 days, during which cells were passaged every 3 days (72 hours). The output cells were counted by a hemacytometer (Hausser Scientific, PA). shows that the number of cells output after the first passage was smaller than at the following times (2nd through 8th passages). This is because cells initially need time to settle down and attach to the surface before they begin to grow. Consequently, cells grow for only part of the first 72 hours. After first passage, cells no longer need time to resettle on the surface. Cells thus have a full 72 hours to grow, leading to greater numbers of output cells than obtained from the first passage. More cells could be easily obtained by adjusting chamber sizes.