- Email: [email protected]
Micropatterned matrigel track for 3D single breast cancer cell metastasis
Abstracts / Journal of Biotechnology 136S (2008) S118–S125
S119
stimulate groups were maintained in culture for 10 days and at the conclusion of the culture period chondrocytes were assayed for the total DNA content, morphology, and cartilage specific gene expression by RT-PCR. Our results show that chondrocytes when stimulated with continuous US for predetermined time intervals, possessed 1.2–1.4 times higher cellular viability than control and had higher levels of type-II collagen and aggrecan mRNA expression when compared to controls1 . In the present study the impact of US stimulation on the morphology, ultrastructure and the cytoskeletal organization of chondrocytes maintain in 3D scaffolds, has now been evaluated. Results depicted in Fig. 1 show that chondroytes remain spherical after US stimulation, with an increased deposition of matrix upon US stimulation and this is consistent with the SEM micrographs of normal chondrocytes. The cytoskeletal elements under US stimulation were visualized by immunofluorescence analysis, and a few selected panels are shown in Fig. 2. Results show that in control, non-stimulated chondrocytes, actin was distributed throughout the cytoplasm and long fibers were observed (panel A), Upon US stimulation, we observed that the organization of actin fibers was redistributed, actin formed a cortical ring, long fibers were absent in most panels (panel B). Interestingly, when ROCK inhibitor was included, actin was diffuse and punctuate and fibers were not observed (panel C). We have observed that in control cells, nuclei were ellipsoidal and possessed the highest fluorescent intensity (panel A). Upon US stimulation, nuclei were still ellipsoidal with lighter fluorescent intensity when compared to control. Based on our results, we suggest that US stimulation alters nuclear shape, perhaps the nuclear architecture, cytoskeletal organization and gross cellular morphology.
parametric studies of cell responses as a function of dispensing pressures and nozzle tip diameters (Chang et al., 2008). To quantify live, apoptotic and necrotic cells as a function of the mechanical perturbations induced by the process parameters, we treated samples with Annexin V staining kit following manufacturer’s protocol. To visualize morphological changes in the nucleus and membrane induced by the system parameters, the cells were also fluorescently labeled with nuclear and plasma membrane stains. The results suggest that the dispensing pressure may produce more affect on cell viability compared to nozzle diameter. Statistical analyses further suggested that the percentage of live, dead and apoptotic cells at statistically significant levels P < 0.05 were quite different for different combination of pressure and nozzle diameter. The results of the study may help to understand the effect of cell injury and the optimization of bioprinting processes.
doi:10.1016/j.jbiotec.2008.07.248
II1-O-009
II1-O-004
Micropatterned matrigel track for 3D single breast cancer cell metastasis
Characterization of cell apoptosis and injury induced by bioprinting process Kalyani Nair 1 , Milind Gandhi 1 , Kenneth Marcolongo 3 , Wei Sun 1,∗
Barbee 2 , Michele
1 Department of Mechanical Engineering and Mechanics, Drexel University, Philadelphia 19104, USA 2 School of Biomedical Engineering, Drexel University, Philadelphia 19104, USA 3 Department of Material Science and Engineering, Drexel University, Philadelphia 19104, USA
E-mail address: [email protected] (W. Sun). Employing cells or other bioactive compounds as basic building blocks to “bio-manufacture” cell-integrated biological models offers tremendous opportunities for regenerative tissue substitutes, physiological and pharmacokinetic models. Bioprinting is one of the enabling biomanufacturing processes that assemblies living cells through mechanical means. Typically, the process consists of simultaneous deposition of cells, biomaterial and/or growth factors under pressure through a micro-scale size nozzle. It is essential to understand the cell responses as well as the subsequent behaviour to the process-induced mechanical disturbances. This paper reports our preliminary studies on characterization of cell responses and injury as a function of bioprinting process. We first introduce the development of a proprietary freeform fabrication based cell dispensing system (Khalil and Sun, 2007), then report
∗ Corresponding author. Tel.: +1 215 8956688; fax: +1 215 8951478.
Acknowledgement Support from National Science Foundation grant (NSF-0700405) to this research is acknowledged. References Chang, R., Nam, J., Sun, W., 2008. Effects of dispensing pressure and nozzle diameter on cell survival from solid freeform fabrication-based direct cell writing. Tissue Eng. 14 (1), 41–48. Khalil S., Sun, W., 2007. Biopolymer deposition for freeform fabrication of hydrogel tissue constructs. Mater. Sci. Eng. C27, 469, 136-13.
doi:10.1016/j.jbiotec.2008.07.249
Temitope Sodunke 1,∗ , Mauricio Reginato 2 , Hongseok “Moses” Noh 1 1
Department of Mechanical Engineering and Mechanics, Drexel University, Philadelphia, PA 19104, USA 2 Department of Molecular Biology and Biochemistry, Drexel University College of Medicine, Philadelphia, PA 19102, USA E-mail address: [email protected] (T. Sodunke). The ability of a cancer cell to disseminate from its tissue of origin and invade other tissues, a process known as metastasis, is the cause of 90% of cancer related deaths (Mehlen and Puisieux, 2006). Despite its clinical significance, only limited information is currently available regarding the mechanism of cancer cell invasion and factors influencing such aberrant behavior. This paper presents an alternative approach to studying breast cancer migration and invasion using a novel microfabricated platform. Micropatterns of matrigel (a widely used biomatrix hydrogel for three-dimensional (3D) epithelial culture) were created using soft lithography techniques such as microtransfer molding. Single fluorescently labeled metastatic breast cancer cells (MDA-MB-231) were placed on one end of the micropatterned track and subjected to a concentration gradient of epithelial growth factor (EGF). We successfully observed the directed movement of the cells towards an increasing concentration of EGF and measured cell migration velocities ranging from 0.2 to 0.6 m/min. Moreover, by embedding the cells within the micropatterned gel, we observed the invasion of cells through the gel layer and damages to the matrigel structure following invasion. We believe that this platform can enable real-time visualization and quantitative measurement of single cell movement, while providing easy parallelization for high-throughput studies.
S120
Abstracts / Journal of Biotechnology 136S (2008) S118–S125
Reference
II1-O-017
Mehlen, P., Puisieux, A., 2006. Metastasis: a question of life or death. Nat. Rev. 6, 449–458.
Dielectrophoretic neuron trapping on semiconductor chips for the extracellular detection of the neuronal network activity
doi:10.1016/j.jbiotec.2008.07.250
P.J. Koester, C. Tautorat, J. Sakowski, W. Baumann, J. Gimsa ∗ University Rostock, Department for Biophysics, Rostock, Germany
II1-O-010
E-mail address: [email protected] (J. Gimsa).
Micropatterned assay plate for three-dimensional high-content hepatotoxicity screening
When cells are seeded on a sensor chip, they are homogeneously distributed on the chip surface. Additional methods have to be applied if single cells, cell aggregates or networks are cultured in a predetermined way on artificial substrates. One can use photolithography (Wyart et al., 2005) or dielectrophoresis (Gimsa, 2001) among others to locate cells on a specific adhesive site or on electrically conducting materials in order to increase the number of located cells. Dielectrophoresis (DP) is the translation of uncharged particles due to the polarization induced by a nonuniform AC field (Gimsa, 2001). In this paper, we apply positive DP in a silicon sensor chip to increase the number of neurons on the electrodes in order to improve the signal yield. Unfortunately, neurons tend to migrate, which lowers the cell number on the electrodes after DP allocation. In future, we want to immobilize cells on the electrodes by, e.g., linking a biochemical matrix. Here, silicon chips contain an array of plane microelectrodes for the measurement of extracellular potentials from neurons, whereas the attracting electrodes are also used as measuring electrodes. Our new neurochip (Ju et al., 2007) features a passive MEA of 58 microelectrodes for the acquisition of action potentials. MEA coating and preparation of cortex tissue from mouse embryo was carried out in saline buffer under sterile conditions. Neuronal signal quality depends on the neuron–electrode coupling properties. The chip quality was controlled by a function generator signal before use. Cell/buffer solution was filled into the chip trough and the DP voltage was applied instantaneously to prevent cell attachment at undesired locations. The used cell solution volume corresponded to approximately 2 × 103 neuronal cells on the MEA. After positive DP and cell attachment, the underlying buffer was removed by a pipette and carefully substituted with supplemented DMEM. In preliminary immobilization experiments, primary neurons were cultured in the presence of salmon fibrin (Ju et al., 2007) in flasks. Localization of the suspended cells on the attracting electrodes is observable. Cells build star like formations around the electrodes. Cells tend to migrate and to move away from the electrodes after some days. Electrophysiological signal acquisition of dielectrophoretically treated chips is demonstrated. For a first experiment with salmon fibrin, primary neurons were overlaid with a fibrin network and survive for at least 5 days. Compared to normal culture, neurons overlaid with fibrin show limited cell motility on the polystyrene substrate. Further investigations will be presented in the full paper (in preparation). DP on silicon sensor chips is a simple method to increase the number of cells at a specific electrode site. A significant improvement of the signal yield should be possible. However, the tendency of the cells to migrate is a problem. Therefore, we suggest using high viscosity molecules or molecular networks by salmon fibrin to immobilize cells at the electrodes.
Edward Keough 1,3,∗ , Peter Clark 1 , Andrew Mastrosante 1 , Brendan Davis 1 , Temitope R. Sodunke 2 , Bonnie J. Howell 3 , Hongseok “Moses” Noh 2 1 School of Biomedical Engineering, Science and Health Systems, Drexel University, Philadelphia, PA 19104, USA 2 Department of Mechanical Engineering and Mechanics, College of Engineering, Drexel University, Philadelphia, PA 19104, USA 3 Department of RNA Therapeutics, Merck Research Laboratories, West Point, PA 19486, USA
E-mail address: [email protected] (E. Keough). Traditional cell-based hepatotoxicity studies performed in vitro in two-dimensional (2D) format have proven unreliable for accurately predicting in vivo responses to drugs. Image-based high-content screening (HCS) in high-throughput screening (HTS) format has shown significant promise over the past decade for achieving increased in vivo correlation (O’Brien et al., 2006). Additionally, three-dimensional (3D) cell culture models will more accurately reproduce the in vivo functions of cellular systems, but these assay devices tend to have significant limitations for integrating with highly automated HCS and HTS machinery. To address this issue, we have utilized soft-lithography micropatterning techniques (Sodunke et al., 2007) to incorporate an organized array of polydimethylsiloxane microwells of 200 m diameter and depth into a 96-well assay plate to support 3D cell growth. Microwells such as these have the capability of generating 3D cultures of human hepatoma (HepG2) cells in static suspension with controllable sizes, producing highly homogeneous cultures. Control over size, position, biological homogeneity and the number of 3D cell cultures is necessary to meet HCS/HTS assay requirements. We have demonstrated the capability of our device by forming spontaneously organized 3D HepG2 cultures through suspension seeding and imaging on a PerkinElmer Opera automated confocal microscope. We believe that by combining 3D cell culture and HCS/HTS technologies, this platform will provide a model that more accurately correlates in vitro results of hepatotoxicity assays to true in vivo conditions. References O’Brien, P., Irwin, W., Diaz, D., Howard-Cofield, E., Krejsa, C., Slaughter, M., Gao, B., Kaludercic, N., Angeline, A., Bernardi, P., Brain, P., Hougham, C., 2006. High concordance of drug-induced human hepatotoxicity with in vitro cytotoxicity measured in a novel cell-based model using high content screening. Arch. Toxicol. 80, 580–604. Sodunke, T., Turner, K., Caldwell, S., McBride, K., Reginato, M., Noh, H., 2007. Micropatterns of matrigel for three-dimensional epithelial cultures. Biomaterials 28, 4006–4016.
doi:10.1016/j.jbiotec.2008.07.251
References Gimsa, J., 2001. Characterization of particles and biological cells by ACelectrokinetics. In: Delgado, A.V. (Ed.), Interfacial Electrokinetics and Electrophoresis. Marcel Dekker Inc., New York, pp. 369–400. Ju, Y.E., Jamney, P.A., McCormick, M.E., Sawyer, E.S., Flanagan, L.A., 2007. Enhanced neurite outgrowth from mammalian neurons in three-dimensional salmon fibrin gels. Biomaterials 28 (12), 2097–2108.
S119
stimulate groups were maintained in culture for 10 days and at the conclusion of the culture period chondrocytes were assayed for the total DNA content, morphology, and cartilage specific gene expression by RT-PCR. Our results show that chondrocytes when stimulated with continuous US for predetermined time intervals, possessed 1.2–1.4 times higher cellular viability than control and had higher levels of type-II collagen and aggrecan mRNA expression when compared to controls1 . In the present study the impact of US stimulation on the morphology, ultrastructure and the cytoskeletal organization of chondrocytes maintain in 3D scaffolds, has now been evaluated. Results depicted in Fig. 1 show that chondroytes remain spherical after US stimulation, with an increased deposition of matrix upon US stimulation and this is consistent with the SEM micrographs of normal chondrocytes. The cytoskeletal elements under US stimulation were visualized by immunofluorescence analysis, and a few selected panels are shown in Fig. 2. Results show that in control, non-stimulated chondrocytes, actin was distributed throughout the cytoplasm and long fibers were observed (panel A), Upon US stimulation, we observed that the organization of actin fibers was redistributed, actin formed a cortical ring, long fibers were absent in most panels (panel B). Interestingly, when ROCK inhibitor was included, actin was diffuse and punctuate and fibers were not observed (panel C). We have observed that in control cells, nuclei were ellipsoidal and possessed the highest fluorescent intensity (panel A). Upon US stimulation, nuclei were still ellipsoidal with lighter fluorescent intensity when compared to control. Based on our results, we suggest that US stimulation alters nuclear shape, perhaps the nuclear architecture, cytoskeletal organization and gross cellular morphology.
parametric studies of cell responses as a function of dispensing pressures and nozzle tip diameters (Chang et al., 2008). To quantify live, apoptotic and necrotic cells as a function of the mechanical perturbations induced by the process parameters, we treated samples with Annexin V staining kit following manufacturer’s protocol. To visualize morphological changes in the nucleus and membrane induced by the system parameters, the cells were also fluorescently labeled with nuclear and plasma membrane stains. The results suggest that the dispensing pressure may produce more affect on cell viability compared to nozzle diameter. Statistical analyses further suggested that the percentage of live, dead and apoptotic cells at statistically significant levels P < 0.05 were quite different for different combination of pressure and nozzle diameter. The results of the study may help to understand the effect of cell injury and the optimization of bioprinting processes.
doi:10.1016/j.jbiotec.2008.07.248
II1-O-009
II1-O-004
Micropatterned matrigel track for 3D single breast cancer cell metastasis
Characterization of cell apoptosis and injury induced by bioprinting process Kalyani Nair 1 , Milind Gandhi 1 , Kenneth Marcolongo 3 , Wei Sun 1,∗
Barbee 2 , Michele
1 Department of Mechanical Engineering and Mechanics, Drexel University, Philadelphia 19104, USA 2 School of Biomedical Engineering, Drexel University, Philadelphia 19104, USA 3 Department of Material Science and Engineering, Drexel University, Philadelphia 19104, USA
E-mail address: [email protected] (W. Sun). Employing cells or other bioactive compounds as basic building blocks to “bio-manufacture” cell-integrated biological models offers tremendous opportunities for regenerative tissue substitutes, physiological and pharmacokinetic models. Bioprinting is one of the enabling biomanufacturing processes that assemblies living cells through mechanical means. Typically, the process consists of simultaneous deposition of cells, biomaterial and/or growth factors under pressure through a micro-scale size nozzle. It is essential to understand the cell responses as well as the subsequent behaviour to the process-induced mechanical disturbances. This paper reports our preliminary studies on characterization of cell responses and injury as a function of bioprinting process. We first introduce the development of a proprietary freeform fabrication based cell dispensing system (Khalil and Sun, 2007), then report
∗ Corresponding author. Tel.: +1 215 8956688; fax: +1 215 8951478.
Acknowledgement Support from National Science Foundation grant (NSF-0700405) to this research is acknowledged. References Chang, R., Nam, J., Sun, W., 2008. Effects of dispensing pressure and nozzle diameter on cell survival from solid freeform fabrication-based direct cell writing. Tissue Eng. 14 (1), 41–48. Khalil S., Sun, W., 2007. Biopolymer deposition for freeform fabrication of hydrogel tissue constructs. Mater. Sci. Eng. C27, 469, 136-13.
doi:10.1016/j.jbiotec.2008.07.249
Temitope Sodunke 1,∗ , Mauricio Reginato 2 , Hongseok “Moses” Noh 1 1
Department of Mechanical Engineering and Mechanics, Drexel University, Philadelphia, PA 19104, USA 2 Department of Molecular Biology and Biochemistry, Drexel University College of Medicine, Philadelphia, PA 19102, USA E-mail address: [email protected] (T. Sodunke). The ability of a cancer cell to disseminate from its tissue of origin and invade other tissues, a process known as metastasis, is the cause of 90% of cancer related deaths (Mehlen and Puisieux, 2006). Despite its clinical significance, only limited information is currently available regarding the mechanism of cancer cell invasion and factors influencing such aberrant behavior. This paper presents an alternative approach to studying breast cancer migration and invasion using a novel microfabricated platform. Micropatterns of matrigel (a widely used biomatrix hydrogel for three-dimensional (3D) epithelial culture) were created using soft lithography techniques such as microtransfer molding. Single fluorescently labeled metastatic breast cancer cells (MDA-MB-231) were placed on one end of the micropatterned track and subjected to a concentration gradient of epithelial growth factor (EGF). We successfully observed the directed movement of the cells towards an increasing concentration of EGF and measured cell migration velocities ranging from 0.2 to 0.6 m/min. Moreover, by embedding the cells within the micropatterned gel, we observed the invasion of cells through the gel layer and damages to the matrigel structure following invasion. We believe that this platform can enable real-time visualization and quantitative measurement of single cell movement, while providing easy parallelization for high-throughput studies.
S120
Abstracts / Journal of Biotechnology 136S (2008) S118–S125
Reference
II1-O-017
Mehlen, P., Puisieux, A., 2006. Metastasis: a question of life or death. Nat. Rev. 6, 449–458.
Dielectrophoretic neuron trapping on semiconductor chips for the extracellular detection of the neuronal network activity
doi:10.1016/j.jbiotec.2008.07.250
P.J. Koester, C. Tautorat, J. Sakowski, W. Baumann, J. Gimsa ∗ University Rostock, Department for Biophysics, Rostock, Germany
II1-O-010
E-mail address: [email protected] (J. Gimsa).
Micropatterned assay plate for three-dimensional high-content hepatotoxicity screening
When cells are seeded on a sensor chip, they are homogeneously distributed on the chip surface. Additional methods have to be applied if single cells, cell aggregates or networks are cultured in a predetermined way on artificial substrates. One can use photolithography (Wyart et al., 2005) or dielectrophoresis (Gimsa, 2001) among others to locate cells on a specific adhesive site or on electrically conducting materials in order to increase the number of located cells. Dielectrophoresis (DP) is the translation of uncharged particles due to the polarization induced by a nonuniform AC field (Gimsa, 2001). In this paper, we apply positive DP in a silicon sensor chip to increase the number of neurons on the electrodes in order to improve the signal yield. Unfortunately, neurons tend to migrate, which lowers the cell number on the electrodes after DP allocation. In future, we want to immobilize cells on the electrodes by, e.g., linking a biochemical matrix. Here, silicon chips contain an array of plane microelectrodes for the measurement of extracellular potentials from neurons, whereas the attracting electrodes are also used as measuring electrodes. Our new neurochip (Ju et al., 2007) features a passive MEA of 58 microelectrodes for the acquisition of action potentials. MEA coating and preparation of cortex tissue from mouse embryo was carried out in saline buffer under sterile conditions. Neuronal signal quality depends on the neuron–electrode coupling properties. The chip quality was controlled by a function generator signal before use. Cell/buffer solution was filled into the chip trough and the DP voltage was applied instantaneously to prevent cell attachment at undesired locations. The used cell solution volume corresponded to approximately 2 × 103 neuronal cells on the MEA. After positive DP and cell attachment, the underlying buffer was removed by a pipette and carefully substituted with supplemented DMEM. In preliminary immobilization experiments, primary neurons were cultured in the presence of salmon fibrin (Ju et al., 2007) in flasks. Localization of the suspended cells on the attracting electrodes is observable. Cells build star like formations around the electrodes. Cells tend to migrate and to move away from the electrodes after some days. Electrophysiological signal acquisition of dielectrophoretically treated chips is demonstrated. For a first experiment with salmon fibrin, primary neurons were overlaid with a fibrin network and survive for at least 5 days. Compared to normal culture, neurons overlaid with fibrin show limited cell motility on the polystyrene substrate. Further investigations will be presented in the full paper (in preparation). DP on silicon sensor chips is a simple method to increase the number of cells at a specific electrode site. A significant improvement of the signal yield should be possible. However, the tendency of the cells to migrate is a problem. Therefore, we suggest using high viscosity molecules or molecular networks by salmon fibrin to immobilize cells at the electrodes.
Edward Keough 1,3,∗ , Peter Clark 1 , Andrew Mastrosante 1 , Brendan Davis 1 , Temitope R. Sodunke 2 , Bonnie J. Howell 3 , Hongseok “Moses” Noh 2 1 School of Biomedical Engineering, Science and Health Systems, Drexel University, Philadelphia, PA 19104, USA 2 Department of Mechanical Engineering and Mechanics, College of Engineering, Drexel University, Philadelphia, PA 19104, USA 3 Department of RNA Therapeutics, Merck Research Laboratories, West Point, PA 19486, USA
E-mail address: [email protected] (E. Keough). Traditional cell-based hepatotoxicity studies performed in vitro in two-dimensional (2D) format have proven unreliable for accurately predicting in vivo responses to drugs. Image-based high-content screening (HCS) in high-throughput screening (HTS) format has shown significant promise over the past decade for achieving increased in vivo correlation (O’Brien et al., 2006). Additionally, three-dimensional (3D) cell culture models will more accurately reproduce the in vivo functions of cellular systems, but these assay devices tend to have significant limitations for integrating with highly automated HCS and HTS machinery. To address this issue, we have utilized soft-lithography micropatterning techniques (Sodunke et al., 2007) to incorporate an organized array of polydimethylsiloxane microwells of 200 m diameter and depth into a 96-well assay plate to support 3D cell growth. Microwells such as these have the capability of generating 3D cultures of human hepatoma (HepG2) cells in static suspension with controllable sizes, producing highly homogeneous cultures. Control over size, position, biological homogeneity and the number of 3D cell cultures is necessary to meet HCS/HTS assay requirements. We have demonstrated the capability of our device by forming spontaneously organized 3D HepG2 cultures through suspension seeding and imaging on a PerkinElmer Opera automated confocal microscope. We believe that by combining 3D cell culture and HCS/HTS technologies, this platform will provide a model that more accurately correlates in vitro results of hepatotoxicity assays to true in vivo conditions. References O’Brien, P., Irwin, W., Diaz, D., Howard-Cofield, E., Krejsa, C., Slaughter, M., Gao, B., Kaludercic, N., Angeline, A., Bernardi, P., Brain, P., Hougham, C., 2006. High concordance of drug-induced human hepatotoxicity with in vitro cytotoxicity measured in a novel cell-based model using high content screening. Arch. Toxicol. 80, 580–604. Sodunke, T., Turner, K., Caldwell, S., McBride, K., Reginato, M., Noh, H., 2007. Micropatterns of matrigel for three-dimensional epithelial cultures. Biomaterials 28, 4006–4016.
doi:10.1016/j.jbiotec.2008.07.251
References Gimsa, J., 2001. Characterization of particles and biological cells by ACelectrokinetics. In: Delgado, A.V. (Ed.), Interfacial Electrokinetics and Electrophoresis. Marcel Dekker Inc., New York, pp. 369–400. Ju, Y.E., Jamney, P.A., McCormick, M.E., Sawyer, E.S., Flanagan, L.A., 2007. Enhanced neurite outgrowth from mammalian neurons in three-dimensional salmon fibrin gels. Biomaterials 28 (12), 2097–2108.