Figure 1.
A worm-cell coculture device for studying nematode migration and behavior in a multicellular microenvironment.
(A) A rendering of the aluminum mold used to cast the PDMS device (B) A rendering of the PDMS-based coculture device consisting of seven parallel lanes. (C) A top-view schematic showing the various regions discussed in this study. Each cell region occupies approximately 25% of the total lane area. The ‘humidifier’ is filled with water in order to limit media evaporation, flow lanes are included in the device for future flow-related experiments but were not utilized in the current study. (D) LECs and HDFs cultured in the device before and after a 3-hour exposure time to the worm (blue = nucleus, green = Actin). Spreading of the cells was less pronounced than what would be seen on polystyrene plates due to the surface roughness of the machine PDMS mold. The presence of the worms in the device did not seem to affect cell viability.
Figure 2.
Block diagram of the worm tracking and thrashing algorithms.
(A) The procedural steps involved in the worm tracking algorithm. Initial worm location in each lane is determined manually. The program then starts the process of acquiring video and cycles through all the lanes by moving to the last known worm location. If the worm is not found, then a linear scanning process initiates in order to find the worm. The scanning process alternates the start position, hence the direction of movement, in order to negate the effect of the microscope stage movement on worm displacement. (B) The thrashing algorithm takes two consecutive images and subtracts them to remove both background and all static features. The resulting image represents degree of worm movement during the time period separating the two frames (∼66 ms for a frame rate of 15 fps). The segment is then thresholded and the mean intensity of the resulting image calculated. The mean intensity is summed for the entire length of an imaging cycle (2 seconds) and the resulting values are normalized to obtain the ‘thrashing index’ metric.
Figure 3.
Tetramisole reduces both worm speed and thrashing.
Representative speed (A) and thrashing (B) over a 3-hour experimental period for three worms under different concentrations of tetramisole (0.0, 1.2 and 2.4 mM) as measured with our platform. Tetramisole is a known paralytic agent that affects nematode thrashing. The gray interval represents a 10-minute gap when the drug was added and imaging session restarted. Our platform can detect changes in both worm speed (C) and thrashing (D) after drug administration. While both speed and thrashing decreased as tetramisole concentration was increased, the thrashing metric was more sensitive to the changes in tetramisole concentration. N = 9, error bars represent standard deviation. Sample videos provided in supplemental materials (S1, S2 and S3).
Figure 4.
L3 B. malayi speed and thrashing, over a 3-hour experimental session, remain constant.
(A–H) Speed and thrashing plots over a 3-hour period under each lane condition. 1) Absence of cells: no cells (nor collagen coating) present in the lane, 2) LECs vs. no cells: LECs on one well and collagen coating on the other, 3) HDFs vs. no cells: HDFs on one well and collagen coating on the other, and 4) HDFs vs. LECs: HDFs on one well and LECs on another. A relatively flat trend was seen for all cases indicating worms were viable and showed consistent behavior throughout the experimental time-frame. N≥28, error band represents standard deviation.
Figure 5.
L3 B. malayi exhibit increased speed and thrashing in the presence of cells.
Average speed (A) and trashing (B) of the worms under different conditions: When there are 1) only LECs in the lane 2) only HDFs in the lane 3) both HDFs and LECs in the lane, and 4) no cells in the lane. The worms were most active when in the LEC lane. They were also more active when only one of the cell types was present compared to both being present in the same lane. N≥28, error bars represent standard deviation.
Figure 6.
L3 B. malayi speed and thrashing are independent of physical contact with cells.
Speed of worms and thrashing behavior when in physical contact with LECs (A, E), HDFs (B, F), HDFs + LECs (C, G) and combined data (D, H). No statistical differences were observed. N≥28, error bars represent standard deviation.
Figure 7.
L3 B. malayi speed and thrashing are correlated in an empty lane, but not when cells are present.
(A) In the empty lanes thrashing correlated with speed (Pearson correlation coefficient; r = 0.81). (B–D) There was no correlation when there were cells in the lane (r = 0.006, −0.049 and 0.12 respectively) which covered 25% of the total lane area. N≥28.
Figure 8.
No difference in percentage of time spent by L3 B. malayi in each lane region.
(A–D) Cell (HDF or LEC) and no cell (only collagen coating) areas each cover 25% of the lane, while the empty region is 50% of the area. No statistical differences were observed when accounting for area differences. N ≥ 28, error bars represent standard deviation.
Figure 9.
L3 B. malayi do not show targeted migration towards LECs or HDFs.
(A) The persistence ratio calculated over the entire time of the experiment (3 hours). (B–E) The persistence ratio calculating for a 10 minute non-overlapping sliding window. (F) A representative velocity plot for one worm illustrating the randomness in directionality. N≥28, error bars and bands represent standard deviation.