Abstract
In this paper we present a microfluidic system based on transparent biocompatible polymers with a porous membrane as substrate for various cell types which allows the simulation of various physiological barriers under continuous laminar flow conditions at distinct tunable shear rates. Besides live cell and fluorescence microscopy, integrated electrodes enable the investigation of the permeability and barrier function of the cell layer as well as their interaction with external manipulations using the Electric Cell-substrate Impedance Sensing (ECIS) method.
1 Introduction
In the body there are various physiological barriers which separate the blood circulation from organs or from the surrounding tissue to block pathogens or tumor cells or to enable a controlled exchange of molecules. Examples are the blood or lymph vessels, the blood-brain barrier, which separates the central nervous system from the blood circulation or the blood cerebrospinal fluid barrier, which demarcates the liquor system of the brain against the blood circulation. In many cases, these barriers are formed by endothelial cells. In vivo studies of these barriers are very difficult and only can be carried out in animal experiments. Therefore we are looking for alternative in vitro testing methods.
Photograph of the microfluidic chip.
Previous transmigration chambers only allow the use under static conditions neglecting the influence of the microcirculation during the physiological processes to be investigated. For that reason we have developed a microfluidic chip system (Fig. 1) which allows the simulation of various physiological barriers under flow conditions. The system consists of two layers with integrated channel systems, separated by a porous membrane. The polymer-based layers are fabricated by hot embossing and connected to each other by thermal bonding. The fluidic ports are mounted using adhesive processes [1].
In order to mimic the physiological barrier one or both sides of the membrane are used as substrate for the corresponding cells (Fig. 2).
Cross section of the microfluidic chip system.
The use of transparent biocompatible materials allows for microscopic in vitro long time observation of living cells. Additionally gold electrodes are integrated on top of the membrane using UV lithography and wet chemical etching in order to perform electrical impedance measurements using the Electric Cell-substrate Impedance Sensing (ECIS) method [2]. Hereby we investigate the permeability and the barrier function of the cell layer [2–6] as well as their interaction with external manipulations (Fig. 3) [7].
Photograph of the microfluidic chip with integrated electrodes on the membrane.
2 Experiments
Up to now the microfluidic chip was used in two applications:
– Examination of the migration process of cancer cells in the microvascular system using the microfluidic chip system as an artificial blood capillary vessel.
– Investigation of neurological diseases using the microfluidic chip system to simulate the blood-brain barrier.
2.1 Microfluidic chip system as an artificial blood capillary vessel
To investigate the migration of cancer cells Human Umbilical Vein Endothelial Cells (HUVEC) are cultivated on top of the porous membrane of the upper channel of the chip system to form an artificial blood vessel. The lower channel represents the surrounding tissue and is filled with tissue like components.
At the beginning human endothelial cells were seeded on the top of the membrane through the upper channel system and cultured in an incubator under constant ambient conditions for at least 24 hours to grow to confluency. Afterwards cancer cells were added to media or to whole blood which was pumped through the upper channel at distinct shear rates, simulating the fluidic conditions of human microvasculature. By means of fluorescence-microscopy it could be shown that a small number of these cancer cells which were stained with calcein green Cell trace, penetrates the endothelial layer and the membrane and finally enters the matrix material of the lower channel [1].
The barrier function of the endothelial layer and their interaction with cancer cells were analyzed by means of Electric Cell-substrate impedance measurement. Figure 5 shows various stages of the cell growth of the endothelial layer on the membrane. The results of the long-term impedance measurements during the whole experiment are shown in Figure 5. At the beginning of the cell seeding, the impedance rises slowly (Fig. 5, I). The reason for this is that the cells impede the electrical path. After 24 hours of growth the cells form a confluent monolayer and the impedance achieved its highest value (Fig. 5, II) [3].
Subsequently, it was observed that the impedance value changes when the cell-cell connection is disturbed by external manipulation, such as interaction by cancer cells (Fig. 5, III) and by trypsin (Fig. 5, IV) which detaches the cells from the surface as shown in Fig. 6.
2.2 Microfluidic chip system as blood brain barrier
For this application first it was tested, which polymer is the best for the new types of cells to be investigated. The selected materials were polycarbonate (PC), which has been used in the artificial blood vessel system and polyethylene terephthalate (PET). In a preliminary test the cell growth and the formation of cell-cell junctions (tight junctions) of human brain microvascular endothelial cells (HBMEC) and the human choroidal plexus papilloma cells (HIBCPP) was investigated on different membranes.
Phase contrast micrographs of different electrodes after 3.5 hours (a) and 24 hours (b) endothelial growth. The formation of an endothelial cell layer (light gray with dark core) on the membrane is characterized by the tight contact of the cells.
Graphs of the impedance.
I - Endothelial cell growth; The impedance increases with the formation of contacts between the cells and between cells and the substrate by the addition of cells;
II - Confluent monolayer after 24 hours; The impedance value reaches its maximum value;
III - Interaction of cancer cells with endothelial cells The impedance value is changed due to cell shape changes;
IV - After addition of trypsin, the cells detach from the surface and the impedance value drops sharply.
Schematic representation of the manipulation of the endothelial cell layer. The tumor cells attach to the cell layer and change its geometry. The cell-cell connection breaks and the tumor cells penetrate the layer (left). After addition of trypsin, the cells detach from the surface and the impedance value drops sharply (right).
Figure 7a shows the results of the staining of the nuclei (blue), ZO1 (red) and the actin cytoskeleton (green) of HBMEC cells on the PC-membrane. We observed only a low expression of ZO1 and cells developed stress fibers (actin staining in green), which indicates that HBMECs do not develop proper barrier functions on the PC membrane. Figure 7b shows the results of staining of the cell nuclei (blue) of HBMEC cells on a PET-membrane. The cells form a dense and uniform layer. In Figure 7c the results of antibody staining against ZO-1 proteins which are significantly involved in the formation of tight junctions on PET are depicted. If successful, the ZO-1 staining forms a red border between the cells. In the second test series with HIBCPP (Fig. 7d, PET) the ZO-1 proteins are stained green. Here too a dense and uniform layer of cells can be detected.
The experiments have shown that both cell types (HBMEC and HIBCPP) form more dense and uniform layers on the PET membranes. For that reason this material is selected for the further investigations.
3 Conclusion
We could show that ECIS measurements enable a detailed characterization of cellular monolayers and therefore allow the investigation of a ripening endothelial cell layer upon shear flow conditions. Furthermore we demonstrated that the impedance of an intact endothelial cell layer is changed by the interplay with stimulating agents and finally by floating and adhering cancer cells. The integration of electrodes for ECIS measurements adds a real-time measurement technique to the microfluidic system next to the previous optical observation methods.
Micrographs of the membrane surface: (a) HBMECs on PC membrane after staining the nuclei (blue), ZO1 (red) and the actincytoskeleton (green). (b) HBMECs on PET membrane after staining the nuclei. (c) PET membrane after ZO-1 staining of HBMEC cells. The arrows point to fully-developed ZO-1 borders around the cells. (d) PET membrane after ZO-1 staining of HIBCPP cells.
For the in vitro simulation of the blood brain barrier it is necessary to colonize the reverse side of the membrane with astrocytes and pericytes to complete the neurovascular unit. The next steps are to investigate the growth of these cell types within the microfluidic system and to verify the reliability of the whole system under flow conditions.
Acknowledgment
This work was partly carried out with the support of the Karlsruhe Nano Micro Facility (KNMF, www.kit.edu/knmf), a Helmholtz Research Infrastructure at Karlsruhe Institute of Technology (KIT, www.kit.edu).
Author's Statement
Conflict of interest: Authors state no conflict of interest.
Material and Methods: Informed consent: Informed consent has been obtained from all individuals included in this study. Ethical approval: The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance the tenets of the Helsinki Declaration, and has been approved by the authors’ institutional review board or equivalent committee.
References
[1] Rajabi T., Huck V., Ahrens R., Apfel M.C., Kim S.E., Schneider S.W., Guber, A.E. Development of a novel two-channel microfluidic system for biomedical applications in cancer research. Biomed Tech. 2012; 57: 921-922.10.1515/bmt-2012-4029Search in Google Scholar
[2] Giaever I., Keese C.R. Micromotion of mammalian cells measured electrically. P.N.A.S. 1991; vol. 88: 7896-7900.10.1073/pnas.88.17.7896Search in Google Scholar PubMed PubMed Central
[3] Keese C.R., Bhawe K., Wegener J., Giaever I. Real-Time Impedance Assay to Follow the Invasive Activities of Metastastic Cells in Culture. BioTechniques 2002; 842–850.10.2144/02334rr01Search in Google Scholar PubMed
[4] De Paola N. Electrical impedance of cultured endothelium under fluid flow. Annals of Biomedical Eng. 2001; 29, 648-656.10.1114/1.1385811Search in Google Scholar PubMed
[5] Sun T. On-ship epithelial barrier function assays using electrical impedance spectroscopy. Lab on a Chip 2010; 10, 1611-1617.10.1039/c000699hSearch in Google Scholar PubMed
[6] Rajabi T., Huck V., Ahrens R., Bassing C., Fauser J., Schneider S.W.. and Guber A.E. Investigation of endothelial growth using a polycarbonate based a microfluidic chip as artificial blood capillary vessel with integrated impedance sensors for application in cancer research. MicroTAS 2013, Freiburg, Germany, 1809-1811.Search in Google Scholar
[7] Rahim S., Üren A. A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells. J. Vis. Exp., 2011.10.3791/2792Search in Google Scholar PubMed PubMed Central
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