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 JoVE Biology

Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors

1, 1, 1, 1, 1, 1

1Institute for Biological Interfaces, Forschungszentrum Karlsruhe

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    Summary

    We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.

    Date Published: 5/21/2008, Issue 15; doi: 10.3791/564

    Cite this Article

    Gottwald, E., Lahni, B., Thiele, D., Giselbrecht, S., Welle, A., Weibezahn, K. Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors. J. Vis. Exp. (15), e564, doi:10.3791/564 (2008).

    Abstract

    We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from non-biodegradable polymers, e.g., polycarbonate or polymethyl methacrylate by micro injection molding, micro hot embossing or micro thermoforming. But, it can also be manufactured from bio-degradable polymers. Its overall dimensions are 0.7 1 x 20 x 20 x 0.7 1 mm (h x w x l). The main features of the chips used are either a grid of up to 1156 cubic micro-containers (cf-chip) each the size of 120-300 x 300 x 300 μ (h x w x l) or round recesses with diameters of 300 μ and a depth of 300 μ (r-chip). The scaffold can house 10 Mio. cells in a three-dimensional configuration. For an optimal nutrient and gas supply, the chip is inserted in a bioreactor housing. The bioreactor is part of a closed steril circulation loop that, in the simplest configuration, is additionaly comprised of a roller pump and a medium reservoir with a gas supply. The bioreactor can be run in perfusion, superfusion, or even a mixed operation mode. We have successfully cultivated cell lines as well as primary cells over periods of several weeks. For rat primary liver cells we could show a preservation of organotypic functions for more than 2 weeks. For hepatocellular carcinoma cell lines we could show the induction of liver specific genes not or only slightly expressed in standard monolayer culture. The system might also be useful as a stem cell cultivation system since first differentiation experiments with stem cell lines were promising.

    Protocol

    This paper describes the use of a chip-based platform (fig. 1) for the three-dimensional cultivation of cell lines as well as primary cells. Since many cells do express organotypic functions only in a 3D-environment, we have developed a polymer chip that provides a scaffold to which the cells can adhere in all spatial directions, and that can be mounted in a bioreactor housing for the control of fluid flow, oxygen tension etc. Depending on the experimental design, the surface of the polymer can be modified by various techniques, e.g., UV-irradiation, PECVD, γ-grafting or conventional wet chemistry.

    564_Figure-01.png

    Figure 01

    1. De-aeration and hydrophilisation of the chip

    Before use, the chip has to be deaerated and hydrophilized. For this, an alcohol series is carried out. Isopropanol solutions consisting of 100%, 70%, 50%, 30% isopropanol in DMPC-treated water are prepared and the chip is dipped in each concentration, beginning with the 100% solution, for up to 30s. The final step of the series consists of pure Dimethyl pyrocarbonate (DMPC)-treated water. From this point on, it is important to keep the chip wet.

    2. Collagen I coating

    After the alcohol series, the chip is usually coated with a collagen I solution from rat tail. From the collagen stock solution of 2 mg/ml in 0.2% acetic acid an aliquot corresponding to 30 μg collagen protein is diluted with DMPC-treated water to a final volume of 150 μl. This results in a collagen coating of the chip surface with a density of 10 μg collagen I per cm2 surface area.

    3. Inoculation of hepatocellular carcinoma cells

    Hepatocellular carcinoma cells of line Hep G2 are trypsinized and counted. For short-term experiments (1 to 6 days) 5*106 cells are inoculated in each chip and the corresponding control 6 cm tissue culture petri dishes. To inoculte, the chip 5*106 cells are resuspended in 150 μl culture medium and placed on top of the microstructured area of the chip (fig. 2). Afterwards, it is placed in an incubator for 2-3 hours. During this incubation period the cells sediment into the micro-containers and adhere to the collagen I-coated scaffold.

    564_Figure-02.png

    Figure 2

    4. Insertion of the chip into the bioreactor housing

    After the incubation period, the chip is removed from the incubator and mounted in the bioreactor housing. For this, under the clean bench, the preassembled bioreactor is removed from the sterile packing and disassembled to a degree that allows for the insertion of the chip. The chip is carefully handled with sterile forceps and placed into the groove that contains the gasket which seals the chip and which results in the generation of an upper and lower compartment in the bioreactor. Then, the bioreactor is assembled again and transferred to the incubator where it is connected to the pump, the gas supply and the oxygen analyser.

    5. Filling of the system

    As soon as the bioreactor is connected to the medium reservoir, pump and gas supply the closed circulation loop is filled with medium. This is done by positioning the 3-way-connectors in such a way that superfusion, which is defined as the flow of medium over the top of the chip, is achieved. This leads to a discharge of enclosed air from the bioreactor circulation without removing the cells from the scaffold. After the system is completely filled with medium, the 3-way-connectors are switched in such a way that perfusion, which is defined as the flow from below the chip through the tissue, is achieved. In the perfusion configuration the flow is adjusted to the cell's needs which for hepatocytes typically ranges from 60-500 μl/min.

    6. Sampling

    During the experiment medium samples can be drawn. For this, syringes are connected to the sterile ports on top of the medium reservoir. After the sampling, the ports are sterilized with 70% isopropanol.

    7. Isolation of intact cells from the chip for downstream applications

    At the end of the experiment, the bioreactors are disconnected from the gas supply and the roller pump, transferred to the clean bench and disassembled as described earlier. With sterile forceps the chip is removed from the bioreactor housing, placed into a 3.5 cm petri dish and rinsed with PBS. Afterwards, the chip is incubated with trypsin/EDTA (0.25%/0.53mM) for 5-15 min in an incubator to detach the cells from the microstructured area. The collected cell suspension is centrifuged for 5 min at 600 g. The cells can then be used for conventional downstream applications, e.g., total RNA or protein isolation. Routinely, we isolate total RNA (PARIS kit, Ambion Inc., Austin, Texas, USA) for microarray analysis and real-time RT-PCR. Protein expression is analysed after immunohistochemical staining of the cells inside the chip with a laser scanning microscope, but can also be analysed otherwise, e.g., by flow cytometry.

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    Discussion

    We have developed a chip-based platform for the three-dimensional cultivation of cells in actively perfused micro bioreactors. The chips can be manufactured from non-biodegradable as well as biodegradable polymers by micro injection molding, hot embossing as well micro thermoforming techniques 3. Depending on the experimental design, the surface of the polymer can be modified by UV-irradiation 4. Hepatocyte cell lines as well as primary rat hepatocytes can successfully be cultivated in these devices as could be shown by expression analysis of some liver specific genes as well as analysis of some liver specific proteins 5,6.

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    Disclosures

    The authors have nothing to disclose.

    Acknowledgements

    We would like to thank Mechthild Herschbach and Anke Dech for excellent technical assistance.

    Materials

    Name Type Company Catalog Number Comments
    Cells Other ATCC HB-8065
    Collagen I from rat tail Reagent Roche Group 11 179 179 001
    PARIS kit Reagent Ambion AM1921
    Syto16 Reagent Invitrogen S7578
    anti cytokeratin 18 Antibody Abcam ab668 Primary Ab, Mouse monoclonal, used 1/100 in PBS
    Anti E-cadherin Antibody Abcam ab1416 Primary Ab, Mouse monoclonal, used 1/50 in PBS.
    Goat anti-albumin Reagent Bethyl Laboratories E80-129 Primary Ab, goat anti-human Albumin, used 1/200 in PBS
    Rabbit anti-mouse IgG1 Antibody Invitrogen A11059 Secondary Ab, Alexa Flour 488 conjugated, used 1/100 in PBS + 0.5 % BSA
    Cy3 anti-goat IgG Reagent Jackson ImmunoResearch 705-165-003 Cy3 AffiniPure donkey a-goat IgG Ab, used 1/700 in PBS + 0.5% BSA

    References

    1. Berry, M.N., Friend, D.S. High-yield preparation of isolated rat liver parenchymal cells: A biochemical and fine structural study. J. Cell Biol. 53, 506-520 (1969)

    2. Seglen, P.O. Preparation of rat liver cells. 3. Enzymatic requirements for tissue dispersion. Exp. Cell Res. 82, 391-398 (1973)

    3. Giselbrecht, S., Gietzelt, T., Gottwald, E., Trautmann, C., Truckenmüller, R., Weibezahn, K.F., Welle, A. 3D tissue culture substrates produced by microthermoforming of pre-processed polymer films. Biomed. Microdev. 8, 191-199 (2006)

    4. Welle, A,, Gottwald, E. UV-based patterning of polymeric substrates for cell culture applications. Biomed. Microdev. 4 (1), 33-41 (2002)

    5. Gottwald, E., Giselbrecht, S., Augspurger, C., Lahni, B., Dambrowsky, N., Truckenmüller, R., Piotter, V., Gietzelt, T., Wendt, O., Pfleging, W., Welle, A., Rolletschek, A., Wobus, A.M., Weibezahn, K.-F. A chip-based platform for the in vitro generation of tissues in three-dimensional organization. Lab Chip 7(6), 777-785 (2007)

    6. Eschbach, E., Chatterjee, S.S., Nöldner, M., Gottwald, E., Dertinger, H., Weibezahn, K.-F., Knedlitschek, G. Microstructured scaffolds for liver tissue with high density: Morphological and biochemical characterization of tissue aggregates. J. Cell. Biochem. 95, 243-255 (2005)

    Comments

    8 Comments

    Hello, I would like to know which culture medium is used for this experiment and which factors for differentiation it contains. If you use the same in flat monolayers cultures , there is  no expression of organotipic functions. Are the Hep G² cells in this system dividing, or only differentiating? And the primary  ones, can they proliferate? Thank you very much
    Reply

    Posted by: AnonymousJuly 14, 2008, 1:46 PM

    Hello, I would like to know which culture medium is used for this experiment and which factors for differentiation it contains. If you use the same in flat monolayers cultures , there is  no expression of organotipic functions. Are the Hep G² cells in this system dividing, or only differentiating? And the primary  ones, can they proliferate? Thank you very much Sorry it was a question
    Reply

    Posted by: AnonymousJuly 14, 2008, 1:46 PM

    Well I suppose it is not possible to proliferate because of the perfusion (and also  hepatic cells don´t  divide very much). I just wondered if it would be possible to get the conditions in the system to get also divisions (with other types of cells) and then differentiation. Thanks
    Reply

    Posted by: AnonymousJuly 14, 2008, 3:25 PM

    Hi, we have published papers from which you can derive that the behaviour of the cells in the bioreactor is dependent on the cell source itself as well as of co-factors. If you use primary cells under differentiation conditions you will get no proliferation and therefore you preserve organotypic functions. In contrast to primary cells, cell lines, like the Hep G² or C3A, will proliferate until the structure is filled and contact inhibition is initiated. During proliferation liver functions of Hep G² and C3A are rather poor. This changes upron reaching confluency in monolayers or reaching a critical cell density in 3D both of which leads to an increase in liver specific parameters expressed by those lines. Besides hepatocytes meanwhile we have cultivated a lot of other cell types in the system, cell lines as well as primary cells. We cannot confirm your statement that we will get no expression of organotypic functions when using the same medium for 3D-culture. On the contrary, exactly because we used the same medium we can prove that changes in the expression patterns are due to the 3D-culture itself. Regards, Eric 
    Reply

    Posted by: AnonymousJuly 17, 2008, 9:23 AM

    Excellent work - are you making this available (how much) ? Mike
    Reply

    Posted by: AnonymousNovember 14, 2008, 11:15 AM

    Hi Mike, thanks for your interest. The problem we have is that we are manufacturing all chips inhouse in relatively small quantities. So before we can bring the system to the market we definitely need an appropriate partner for the manufacturing of the chips in the quality and quantity we need. Since we are using straight forward methods for the manufacturing process that are often not compatible with mass production this results in the necessity of building completely new machins from scratch. Due to the global lack of investments into new technologies this is a very hard business todate. However, the idea of marketing the system keeps us going and I'm sure that you will hear from the system much more, be it commercial or scientific. Regards, Eric
    Reply

    Posted by: AnonymousJanuary 22, 2009, 8:29 AM

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