Cell Culture Biochips for Optical Microscopy

Methods in molecular biology and the understanding of actions inside living cells have changed drastically over the last decade. In comparison, methods in cell culture have remained unchanged for almost a century, when the first type of Petri dish was described.

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

Figure 1 - μ-Slide I and a cell observed with different imaging modes in a cell culture chip for live cell imaging.

The cell culture μ-Slide system (ibidi GmbH, Munich, Germany) is well suited for the cultivation and subsequent high-end optical analysis of cells. In addition to fluorescence applications, the system can be used with phase contrast or differential interference contrast (DIC). The μ-Slide I and a cell observed with different imaging modes are shown in Figure 1.

μ-Slides contain channels and reservoirs to grow the cells on the same substrate where, later, fixation, staining, and imaging are carried out. Thus, there is no need to transfer the cells to a coverslip.

The system is a flow-through device for the functional analysis of living cells. Apart from some open formats, most of the systems are perfusion chambers. They allow easy exchange of fluids and are useful for the cultivation of cells under defined shear stress conditions.

The μ-Slide I was used to cultivate human umbilical vein epithelial cells (HUVECs) under flow conditions. The influence of different coatings and flow rates on cell growth could be observed. A channel fully covered with HUVECs was obtained by sequential seeding steps, providing a model system for blood vessels in general.

Figure 2 - a) Y-shaped μ-Slide with adopted flow system. b) Schematic drawing of an artificial blood vessel in the system.

Another approach utilizing the advantages of a perfusion chamber is the investigation of plaque formation inside an artificial blood vessel. The flow-through device can be easily connected via its LUER adaptors, and the system is ready to be used as a disposable cell culture biochip to meet high optical demands. The main research area here was to study the influence of the velocity gradients at the bifurcation of the vessel. These branching points are of special interest in arteriosclerosis research because the flow conditions change drastically. In an ongoing project, the authors are studying the influence of certain substrates to cell adhesion. At ibidi the authors plan to do a screening to identify which substances start an inflammation process on an endothelial layer at the bifurcation. Additionally, the authors will simulate cell-cell interactions in their blood vessel model systems. Figure 2a shows a Y-shaped μ-Slide together with a diagram to illustrate the flow inside such an artificial blood vessel (Figure 2b).

Due to its ease of handling, the μ-Slide VI flat is suitable for rapid parallel immuno-fluorescence assays, e.g., in surface immobilization, binding, and imaging protocols. In six parallel channels, cells can be seeded in a volume of only 25 μL, while a microscopic area of 100 mm2 per channel is generated. The parallelization enables the easy comparison of different cell lines, fixation, or staining methods on a single slide.

In a 25-μL cell suspension, 10,000 cells per channel can be observed. After cultivation, cells can be fixed and stained. For fixation, all standard methods (i.e., methanol, acetone, and paraformaldehyde) can be used. The plastic material is compatible with all commonly used substances.

Figure 3 - Fluorescence image of cells taken in μ-Slide VI flow.

Figure 4 - Fluorescence image of cells taken in μ-Slide VI flow.

Only 25 μL of staining solution is required. Subsequently, different staining protocols, concentrations of staining solutions, or antibodies can be applied to the same set of cells on one slide. All washing procedures and optimization steps can be performed right under the microscope. The images shown in Figures 3 and 4 can be achieved on a single μ-Slide in almost no time. Due to the small volumes of staining solution required, the costs of such an assay can be reduced by a factor of 10–20.

In addition, the convenience of the μ-Slides is an important advantage. Until now, cells had to be seeded on glass coverslips in a six-well plate, and washing and further treatment were tedious. With μ-Slides, there is no cracking of fragile coverslips, spilling of fluids onto the microscope, contact with organic solvents, or staining solutions. The entire procedure is done in the protected surroundings of the channel. Stringent washing steps are easy and highly reproducible. For each assay, there is a time reduction of approx. 30 min.

Figure 5 - μ-Slide VI flow.

Another important application of μ-Slides is the toxicological screening of chemicals such as potential drug candidates and environmental toxins, and for this, the μ-Slide I and μ-Slide VI flow are particularly well suited. The perfusion chamber permits the easy addition of candidate drugs to adherent cells on the surface. Measurements can be taken with the microscope with a ready-to-use flow kit (see Figure 5). Alternatively, a time-lapse series can be taken and pharmacological screening of substances can be made in long-term studies using the perfusion chambers. Consequently, cell-based assays that require an optical readout can be easily performed in a μ-Slide VI flow. For example, the influence of differently concentrated cadmium solutions on COS cells have been tested (results to be published).

The major focus of ibidi’s research projects is chemotactical assays. An ideal system for these assays is Dictyostelium discoideum. This widespread model system is known to migrate in a cyclic adenosine monophosphate (cAMP) gradient. The channel geometry of μ-Slide I is well suited for these types of assays, in which the chemoatractant is added from one side and the motion of the cells can be observed in a time-lapse series directly under the microscope. (The Dictyostelium discoideum mutant used here [DdLimE-GFP] was a kind gift from Dr. Günther Gerisch, MPI for Biochemistry, Martinsried, Germany.)

Figure 6 - Sequence from a time-lapse series analyzing the Dictyostelium discoideum velocity in a cAMP gradient.

In one set of experiments, 40 μL of a 0.1 mM concentration of cAMP was injected into the channel using a simple pipetting procedure. Subsequently, the motion of the Dictyostelium discoideum was monitored over time (see Figure 6). Most of these social amoebae move over one-third to one-half of the observation window, which is 425 μm wide. Thus, a cell velocity of 5–7 μm/min is observed here, which corresponds to the values reported in the literature.

Outlook

Lab on a Slide® technology (ibidi GmbH) allows the handling and analysis of biological samples. Therefore, cells, bacteria, and viruses can be investigated, immobilized, and manipulated using high-resolution microscopy. This includes confocal, atomic force microscopy (AFM), and FCS measurements. Biocompatible surfaces enable the investigation of drug uptake and drug pathways inside living cells.

Looking to the future, these disposable cell culture chips will be of interest from a cell biological as well as a biophysical point of view. They allow a deeper look at protein interaction, drug uptake, and blood vessel formation. In addition, a better understanding of cell attachment to well-characterized surfaces under defined shear stress will improve the development of new products in the analytical, cosmetic, and pharmaceutical industries.

Dr. Rädler is Head, Chemical Research & Functional Surfaces, and Mr. Horn is Product Manager, Chemotactical Assays, ibidi GmbH, Schellingstrasse 4, 80799 Munich, Germany; tel.: +49 89 2180 6418; fax: +49 89 2180 13539; e-mail:uraedler@ibidi.de.

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