Experimental Video
Time-lapse sequences of phase contrast images showing the motility of fish epidermal keratocyte cells at three different densities. Each video is 4 hours long. Robust collective behavior can be observed as the density of cells reaches a critical value around 5x10-4 cell per square microns. This spectacular ordering phenomenon resembles the well-known flocking of fish or birds.
Simulation Video
Computer simulations were preformed of the system described in the text for different values of the number density, these showed a transition to the ordered phase similar to that seen in experiments. In the second frame, at a density just below critical a complex interplay of interacting groups of cells moving in random directions can be observed.
Direct link to the experimental video (MPEG-2, ~4 MB) is here,videos/experiment.mpgvideos/experiment.mpgshapeimage_1_link_0
Direct link to the simulation video (MPEG-2, ~20 MB) is here.videos/simulation.mpgvideos/simulation.mpgshapeimage_2_link_0
Experimental Details
Time Lapse Microscopy
We used a home developed fully computer-controlled time-lapse microscope, which enabled us to monitor cell motility with a frequency of 1 shot per min in up to four cultures with several fields of view each. Cultures were positioned over the 10x objective of a Leica DM IRB inverted phase contrast microscope by a 2D motorized stage (Marzhauser). Either an Olympus C4040-z digital camera or a DP70 fluorescent camera was used to capture the images. During the typically 24-h long time-lapse microscopy experiments live cells were kept in a custom made room temperature mini-incubator to maintain a 5% CO2 atmosphere and to avoid steaming up.
Cell Cultures
2-4 fish scales were collected from living 5-15 cm goldfish (Carassius auratus) with tweezers, and placed external side up in a 35-mm circular Petri dish (Greiner) similarly to Svitkina et al. (J. Cell Biol. 139 (2), 397). After a few seconds scales were flooded with 1 ml culture medium consisting of RPMI 1640 supplemented with 10% fetal calf serum (Gibco), 40 ug/ml Gentamycin (Gibco) and 0.5 ug/ml Amphotericin B (Sigma). Scales were kept overnight at room temperature in a 5% CO2 atmosphere to allow epidermal keratocyte cells to migrate out from the scales. Before time-lapse microscopy scales were removed, and cultures were flushed with fresh medium. To obtain low density cultures we applied phosphate saline buffer (PBS) for 5-10 min and/or 0.25% Trypsin (Gibco) for 1-2 min. PBS and trypsin reversibly weaken cell-cell and cell-substrate connections, allowing the removal of a controllable percentage of the cells.
Video (.mov, ~25MB) of a small scale simulation showing more detail is here.videos/density.movvideos/density.movshapeimage_3_link_0
Simulation Video of Model Cells in a Square Arena
Computer simulations of model cells confined to a square arena were preformed. These simulations showed the emergence of collective circular motion over a wide range of model parameters.
Direct link to the simulation video (MPEG-2, ~20 MB) is here.videos/many.mpgvideos/many.mpgshapeimage_4_link_0
Experimental Video of Keratocytes in Microfabriceted Arena
Montage of time-lapse sequences obtained from several fields of view show a sustained whirling motion of the cells when confined to a 2X2 mm2 square shaped microfabricated arena.
Direct link to the experimental video (MPEG-2, ~10 MB) is here.videos/keratmedencek04_montage_oraval.mpgvideos/keratmedencek04_montage_oraval.mpgshapeimage_5_link_0
Video (MPEG-2, ~20MB) of a lower density simulation is here.videos/few.mpgvideos/density.movshapeimage_6_link_0