Chemotaxis is an important field in biology today because it plays a crucial role in neovascularization of solid tumours, metastasis of cancer cells as well as wound healing and many immunological processes. Our chemotaxis approach includes the evaluation of a suitable chamber design for chemotaxis measurements of slow migrating adherent cells like cancer and endothelial cells. The fluorescence measurements of the Alexa 488 concentration clearly show that the chamber design and geometry is well-suited for establishing gradients that remain stable over more than 48 hrs, which is unique for static assays of this kind.
By using plugs, the chamber can be completely sealed and pressure differences relax extremely fast in the system. Inside the small structures, the system is characterized by low Reynolds numbers resulting in a purely diffusive formation of the gradient. In the absence of externally applied pressure, no convection or flow appears as the system is dominated by friction forces.
There is, however, a limitation of the chamber, due to the long time required to establish the gradient, as it takes about 8-12 hours to establish a quasi steady state that is characterized by a linear gradient in the region of the observation area. Fortunately, working with HUVEC and HT-1080 cells, we found that reproducible data can be collected starting immediately after preparing the gradient. The time development shown in Figure indicates that after 3 to 4 hours both FMI components attain their final values, whereas the centre of mass immediately starts to move in direction of the chemoattractant source with rather constant velocity. This finding implies that both kinds of cells are able to sense the gradient during its formation. This result seems less spectacular when keeping in mind that cells in which the absolute cell concentration of the immediate surrounding is low are much more sensitive to spatial concentration changes.
The developed preparation protocol makes observation of chemotaxis of HT-1080 and HUVEC possible, each representing either a commonly used cell line or primary cell type. The ability for co-culturing different cell types allows for testing of the attraction of one cell type by molecules secreted by another cell type. The capability to follow cell migration with video microscopy in combination with the introduced "Chemotaxis and Migration Tool" that is freely available for ImageJ provides access to a complete set of characteristic chemotaxis and migration parameters. We characterize migration of cells in 2D by the parameters average cell velocity and directness and chemotaxis by displacement of centre of mass and forward migration indices in directions both parallel and perpendicular to the direction of the gradient, i.e. FMI║ and FMI┴. The "Chemotaxis and Migration Tool" can also be used for various other analysis methods like angle distribution of cell end points, etc.
Our findings show that parameters like the average velocity and the displacement of centre of mass strongly depend on the motility, which is an intrinsic cell property. Only in cases where cells have the same ability to move can parameters be directly compared in a meaningful way. In our example, this condition is not valid for the HUVEC gained from two different sources. We found HUVEC from different sources 1 and 2 migrated with very different average velocities, namely 22.5 μm/hr and 51.1 μm/hr. The first value is nearly in exact agreement with the value of 23 μm/hr given in the literature [27
]. Generally it is expected that chemokines like VEGF used as chemoattractants also have influence on the motility of cells [30
]. From our findings the parameters that appear to be independent with respect to motility include FMI, directness, and the Rayleigh value. Consequently, we conclude that chemotaxis might be best defined through comparison of the parallel FMIs of the Chemotaxis experiment +/- with the perpendicular FMI of +/- and the FMIs of the positive +/+ and negative -/- controls. For basic analysis of chemotaxis, we recommend the use of FMIs and Rayleigh values, as this combination contains sufficient information to characterize chemotaxis. Values like average cell velocity and directness as well as the cell videos allow for detailed observation of the characteristics associated with the movement of cells and cell reaction to reagents. The presence or lack of gradients did not result in any variance in the measured directness of HUVEC cell movement. We therefore conclude that enhancement of directness is not a necessary criterion for evaluation of chemotaxis.
One drawback of video microscopy as a measuring method is the low throughput due to problems that frequently arise when attempting to heavily parallelize the tests. We therefore analyzed the time dependence in order to determine an optimal measurement duration. From Figure , we conclude that for HT-1080, twelve hours should be sufficient to analyze the chemotaxis effect. Plotting the time evolution is an effective tool for optimizing the duration of chemotaxis experiments.
There are several viable methods to measure chemotaxis in vitro
. The Boyden chamber assay is relatively easy to use, requires no expensive equipment and provides quantitative data. Although quantitative assessment of cell migration is possible with a number of methods, the most widely used is the counting of the number of cells that migrate through the filter in several microscopic fields. However, observation of the cells during the migration is not possible, so measurements of cell morphologies and cell speed are not available. The Zigmond-chamber improves on the Boyden design with the decisive advantage of the possibility of microscopy during cell movement accompanied by the disadvantage of complex handling. Consequently, widespread usage is not practical. An easy and economic method is the agarose assay [31
], with which the primary benefit is the possibility to observe the chemotaxis with the naked eye without a microscope. Furthermore, it is possible to establish multiple gradients in a single experiment. However, the time-intensive preparations and the instable and poorly defined time gradients are significant disadvantages.
Nevertheless, the Boyden chamber remains the most widely used of all traditional assays. Its two major drawbacks are instability of gradients and poor control over gradient shape in the vicinity of cells. The local gradient may be controlled by the use of point release of chemokines from micropipettes; however, these are only useful for short examinations of fast moving cells before diffusion degrades the gradient and chemokines accumulate in the environment. In Boyden chamber experiments, adherent slow migrating cells are often seeded out to very high densities such that the pores are closed by the cells and chemoattractant is trapped in the pores at high concentration. The cells experience the full concentration of the chemoattractant in the area pointing into the pore but extremely low chemoattrantant concentration in the rest of the cell membrane area. This condition is an accurate simulation of stepwise chemical gradients, which might appear at vessel walls. In contrast, the μ-Slide Chemotaxis is made for simulating shallow gradients, which might appear in interstitial tissue. In this region gradients are formed through chemoattractant sources such as starving tumour cells or inflamed tissue communicating with other cells over distances on the order of a millimeter. The width of the observation area in between the two reservoirs has been chosen to be one mm so that the relative change in concentration over an estimated cell length of 20 μm is always larger than 2%.
Compared to transwell, agarose, or Zigmond chamber assays, microfluidic systems allow for improved control and linearity of chemokine gradients. Furthermore, microfluidic systems are also designed to form various gradient shapes and spatial gradients of different profiles. On the other hand, traditional assays are still preferred because of their simplicity as well as their support for almost every cell type. Additionally, the presence of flow leads in many microfluidic devices result in some significant limitations, and they cannot be used with cells that are sensitive to shear stress. Finally, the microfluidic systems are not easy to use [18
The new chamber described in this publication is easy to handle, allows for the creation of a stable gradient, and permits up to three independent experiments during one examination. Furthermore, life cell microscopy is feasible so that cell movement, cell morphology and cell interaction are observable. Due to the high optical quality (see Figure ), microscopy measurement with high resolution can also be performed to better evaluate the molecular aspects of cell movement and chemotaxis in chemical gradients.
Figure 8 Single HT-1080 cell, migrating towards a FCS gradient pointing from the right to the left side (higher concentration is on the left side). The image was acquired with an epi fluorescence mode using a 60× oil immersion objective (Nikon). Scale (more ...)