<b>Cell Culture Biochips For Imaging The Directed Motion Of Cancer Cells</b><br/><br/>

Cell Culture Biochips For Imaging The Directed Motion Of Cancer Cells

Dr. Ulf Rädler

Introduction

Recently, there have been major developments in the optical analysis of processes inside living cells. Fluorescence methods, confocal microscopy and evanescent field techniques have become indispensable tools for cell imaging. Consequently, there is an increasing demand for systems combining the needs for cultivation of the cells with the requirements of high-end microscopy.

Figure1. New ibidi μ-Slide Chemotaxis with three parallel chambers for long term chemotaxis assays. The colors indicate either a chemoattractant is present or not.

Therefore, ibidi (Munich, DE and Verona, WI) developed the μ-Slide family as microfluidic carriers, which allow the simultaneous cultivation and optical analysis of cultured cells. The carriers enable immunofluorescence based assays as well as live cell imaging and cell studies under perfusion conditions. In this article we introduce a new chemotaxis μ-Slide that enables chemotactical and migration studies.

Concentration gradients of a large variety of substances induce chemotaxis — the directed motion of cells. Due to its importance for angiogenesis, oncology, neurology, and especially immunology, the question of migration under special stimulation gains a lot of interest. So far, directed migration of cancer cells could not be observed directly under the microscope as the gradients that could be produced have not been stable enough for long term assays. Recently, our research team developed a new μ-Slide for chemotactical analysis (Figure 1) to overcome the disadvantages of existing chemotaxis chambers. The new design allows high resolution microscopy, convenient liquid handling, and live cell imaging under defined linear concentration gradients to work over extended periods of time.

Cell tracking and statistical analysis are powerful tools in migration studies of random and biased walks. Video microscopy allows tracing of adherent moving cells over time. The new chemotaxis system intends to observe slowly migrating cells (cancer cells, endothelial cells) in real time.

Here we describe a first set of data generated using HT 1080 fibrosarcoma cancer cells. The chamber and the analyzed tracking data are presented.

Experimental set-up

Figure 2. Basic principle
of the &#956-Slide Chemotaxis indicated by a schematic cross section. Adherent cells are grown in a small observation slit only and become superimposed
by a linear gradient of chemoattractant. The enormous time stability of &lt48 hours allows observations of even slow cells.

Cell culture. Human fibrosarcoma epithelial cell line HT 1080 was grown in Dulbecco's modified Eagle's Medium (DMEM) supplemented with 10 % fetal calf serum (FCS) at 37 C and 5% CO₂. HT 1080 cells were cultivated to near confluency, trypsinized, and filled into the chemotaxis chambers using 3 μl cell suspension of a density of 3吆⁶ cells/ml. DMEM containing 0.1% bovine serum albumin was used as attractant free medium (C₀). DMEM with 10% FCS was used as chemo-attractant (C₁₀₀). Chemotaxis chambers were coated with Collagen IV and allowed to dry prior use.

Chemotaxis chamber. The chemotaxis chamber is designed as a one-piece system for inverted microscopes (Figure 1). The chamber is made from high optical quality plastic with a coverslip-like bottom (No. 1.5). The design consists of two large reservoirs, 40 μl each, connected by a 75 μm high slit forming the 2ࡧ mm observation area. The cross section of such a system is shown in Figure 2. Two small side channels enable flushing in a cell suspension into the observation area only. Cells growing in this slit become superimposed by a chemo-attractant, which is added into one reservoir via pipette adapter inlets. One slide with standard microscopy format contains three assay chambers allowing parallel observations.

Figure 3. Overlay of a phase contrast image of HT 1080 cells after 12 hours and the cells´ paths over the entire period.

Microscopy. Time lapse video microscopy with cells was performed using an inverted microscope (Zeiss, Axiovert 100, objective 10×), an automated and incubated stage, and a CCD camera. Phase contrast images were taken at 5 minute intervals. Cell tracking was performed using manual tracking software (Figure 3). Tracking results were analyzed and plotted by a self-programmed ImageJ plug-in.

Results and discussion

In general, chemotaxis is the directed motion of cells towards or away from a chemo-attractant. That chemotactical motion was first described by Pfeffer in 1884 /⁽¹⁾ If the cells move randomly, there center of mass is not changed, as indicated in Figure 4a. Therefore, after analyzing the cell traces, migration data showed no significant bias when using no chemo-attractant.

Figure 4. The cells´ paths normalized to a plot and circular counts visualized in a rose diagram. A) Control experiment without significant bias. B) Strong chemotactic effect downwards. Click to enlarge.

By the use of FCS as an attractant a biased walk can be observed (Figure 4b). Analyzing the cells´ paths showed a mean cell speed of 21 μm/hr. The new center of mass (blue cross), after 12 hours of migration, is (x=5 μm, y=㫏 μm) which is a highly significant effect towards the attractant (Rayleigh test, p <0.05). Rose diagrams (Figure 4b) also illustrate strong effects of cell motion in the direction of the chemo-attractant (in this case downwards). This data implies the reliability of the method analyzing chemotaxis of slowly migrating cancer cells. The tested fibrosarcoma cell line HT 1080 is used regularly in chemotactical assays.⁽²⁾

In a typical visual chemotaxis experiment three chambers are required. One for the real experiment, where either a putative factor as attractant, or manipulated cells, are tested for directed migration. Then, two control experiments are necessary; one for a control without any attractant and one with a uniform attractant concentration. Therefore, we designed 3 chemotaxis assay chambers on one single slide (see Figure 1), so one can also test different substances directly on one carrier. Using an automated stage all chambers can be observed in parallel.

So far, the direct observation of chemotactical motion was not easily possible in existing chambers because they are often inconvenient in handling, gradient and time stability. Additionally, glass chambers have to be assembled and cells have to be seeded on coverslips before starting the experiment. On the other hand, those visual assays for video microscopy like the Zigmond chamber⁽³⁾ or the Dunn chamber⁽⁴⁾ provide much more information than the commonly used Boyden chambers⁽⁵⁾ in which cells have to crawl through porous membranes. Those membranes make microscopy impossible and provide information only on either cells are crawling or not.

Figure 5. Time series of a single Dictyostelium discoideum cell moving along a gradient of cAMP recruiting DdLimE-GFP to the leading edge. Click to enlarge.

Directly analyzing cell traces via microscopy one is able to access speed, directionality, turning behavior, cell-cell-interactions and gradient dependencies. The ability to use high-end microscopy combined with cell tracking software allows the user to visualize fluorescent proteins in real time during chemotaxis. Therefore, cell polarization and recruitment of involved proteins can be observed with the described new tool (Figure 5). Additionally, real chemotaxis effects can be distinguished from random or increased migrating behavior in one single experiment.

About the authors

Dr. Ulf Rädler is head, chemical research and functional surfaces. Elias Horn is product manager chemotactical assays. The authors would like to express their thanks to the VDI Technologiezentrum, as this work was partially funded by grant number 13N8777. Please contact Dr. Rädler (uraedler@ibidi.de) for further information.

References

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2. Sloan, K. E. et al. BMC Cancer 4:73 (2004).
3. Zigmond, S. H. J. Cell Biol. 75:606-616 (1977).
4. Zicha, D. J. Cell Sci. 99:769-775 (1991).
5. Boyden, S. J. Exp. Med. 115 (1962).