A multi-inlet microfluidic device fabricated for in situ detection of multiple cytotoxicity endpoints

Abstract

This work describes the development of a multi-inlet microfluidic device for in situ detection of multiple cytotoxicity endpoints. The device consists of an upstream microfluidic multi-inlet module (μMIM) and a downstream microfluidic cell culture channel (μCCC). The integration of a device with syringe pumps via the inlets of MIM mainly enables the performance of multiplex cytotoxicity assays targeted on multiple endpoints. The device in the study was used for the long-term culturing of BALB/3T3 fibroblast cells. In addition, cadmium ion was used as a toxicant to induce the toxicity in the cultured cells. The cytotoxicity endpoints, such as the in situ detection of reactive oxygen species (ROS), nuclear structures and cell morphology, were examined and correlated simultaneously for every single cell after they were exposed to cadmium. The data indicated that the percentage of cells that had produced ROS and shown nuclear staining were slightly higher than the cells that had produced ROS only, but this percentage was significantly higher than the cells that had shown only nuclear staining. The results revealed that the obtained correlated data from simultaneous observations of multiplex cytotoxicity assays were more efficient in providing the mechanistic explanation for Cd-induced toxicity than the individual data that were derived after examining a single endpoint. In addition, the results suggested that the intracellular formation of ROS induced by cadmium might have been followed by the alterations in cell and nuclear morphology and loss of membrane integrity. The device allows for the real-time monitoring of cells and their behavior against a toxicant and facilitates the rapid performance of cytotoxicity testing. Furthermore, we anticipate that the presented device could be very effective in advancing the understanding of the precise mechanism of cytotoxicity by improving the sensitivity and accuracy in identifying endpoints.

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References

  1. 1.

    Beebe, D.J., Mensing, G.A. & Walker, G.M. Physics and applications of microfluidics in biology. Annu. Rev. Biomed. Eng. 4, 261–286 (2002).

    Article  CAS  Google Scholar 

  2. 2.

    Kang, L., Chung, B.G., Langer, R. & Khademhosseini, A. Microfluidics for drug discovery and development: from target selection to product lifecycle management. Drug Discov. Today 13, 1–13 (2008).

    Article  CAS  Google Scholar 

  3. 3.

    Ros, A., Hellmich, W., Regtmeier, J., Duong, T.T. & Anselmetti, D. Bioanalysis in structured microfluidic systems. Electrophoresis 27, 2651–2658 (2006).

    Article  CAS  Google Scholar 

  4. 4.

    Hung, L.-H. & Lee, A.P. Microfluidic devices for the synthesis of nanoparticles and biomaterials. J. Med. Biol. Eng. 27, 1–6 (2007).

    Google Scholar 

  5. 5.

    Sarrazin, F., Salmon, J.-B., Talaga, D. & Servant, L. Chemical reaction imaging within microfluidic devices using confocal raman spectroscopy: the case of water and deuterium oxide as a model system. Anal. Chem. 80, 1689–1695 (2008).

    Article  CAS  Google Scholar 

  6. 6.

    Andersson, H. & van den Berg, A. Microfluidic devices for cellomics: a review. Sens. Actuators B: Chem. 92, 315–325 (2003).

    Article  Google Scholar 

  7. 7.

    Chung, B.G., Lin, F. & Jeon, N.L. A microfluidic multiinjector for gradient generation. Lab Chip 6, 764–768 (2006).

    Article  CAS  Google Scholar 

  8. 8.

    Dai, W., Zheng, Y., Luo, K.Q. & Wu, H. A prototypic microfluidic platform generating stepwise concentration gradients for real-time study of cell apoptosis. Biomicrofluidics 4, 024101 (2010).

    Google Scholar 

  9. 9.

    Kim, M. & Kim, T. Diffusion-based and long-range concentration gradients of multiple chemicals for bacterial chemotaxis assays. Anal. Chem. 82, 9401–9409 (2010).

    Article  CAS  Google Scholar 

  10. 10.

    Niu, X., Gielen, F., Edel, J.B. & de Mello, A.J. A microdroplet diluter for high-throughput screening. Nat. Chem. 3, 437–442 (2011).

    Article  CAS  Google Scholar 

  11. 11.

    Mosadegh, B. et al. Integrated elastomeric components for autonomous regulation of sequential and oscillatory flow switching in microfluidic devices. Nat. Phys. 6, 433–437 (2010).

    Article  CAS  Google Scholar 

  12. 12.

    Spence, D.M. Automation and microfluidic assays: in vitro models of the mammalian microcirculation. JALA 10, 270–275 (2005).

    CAS  Google Scholar 

  13. 13.

    Wang, Z., Kim, M.-C., Marquez, M. & Thorsen, T. High-density microfluidic arrays for cell cytotoxicity analysis. Lab Chip 7, 740–745 (2007).

    Article  CAS  Google Scholar 

  14. 14.

    Poulsen, C.R., Culbertson, C.T., Jacobson, S.C. & Ramsey, J.M. Static and dynamic acute cytotoxicity assays on microfluidic devices. Anal. Chem. 77, 667–672 (2005).

    Article  CAS  Google Scholar 

  15. 15.

    Walker, G.M., Monteiro-Riviere, N., Rouse, J. & O’Neill, A.T. A linear dilution microfluidic device for cytotoxicity assays. Lab Chip 7, 226–232 (2007).

    Article  CAS  Google Scholar 

  16. 16.

    Mahto, S.K., Yoon, T.H., Shin, H. & Rhee, S.W. Multicompartmented microfluidic device for characterization of dose-dependent cadmium cytotoxicity in BALB/3T3 fibroblast cells. Biomed. Microdevices 11, 401–411 (2009).

    Article  CAS  Google Scholar 

  17. 17.

    Ma, B., Zhang, G., Qin, J. & Lin, B. Characterization of drug metabolites and cytotoxicity assay simultaneously using an integrated microfluidic device. Lab Chip 9, 232–238 (2009).

    Article  CAS  Google Scholar 

  18. 18.

    Tourovskaia, A., Figueroa-Masot, X. & Folch, A. Differentiation-on-a-chip: a microfluidic platform for longterm cell culture studies. Lab Chip 5, 14–19 (2005).

    Article  CAS  Google Scholar 

  19. 19.

    Korn, K. & Krausz, E. Cell-based high-content screening of small-molecule libraries. Curr. Opin. Chem. Biol. 11, 503–510 (2007).

    Article  CAS  Google Scholar 

  20. 20.

    Crow, J.P. Dichlorodihydrofluorescein and dihydrorhodamine 123 are sensitive indicators of peroxynitrite in vitro: implications for intracellular measurement of reactive nitrogen and oxygen species. Nitric Oxide 1, 145–157 (1997).

    Article  CAS  Google Scholar 

  21. 21.

    Myhre, O., Andersen, J.M., Aarnes, H. & Fonnum, F. Evaluation of the probes 2′,7′-dichlorofluorescin diacetate, luminol, and lucigenin as indicators of reactive species formation. Biochem. Pharmacol. 65, 1575–1582 (2003).

    Article  CAS  Google Scholar 

  22. 22.

    Shono, M. et al. Differences in ethidium bromide and 4′-6-diamidino-2-phenylindole staining profiles with regard to DNA fragmentation during apoptosis. Biochem. Mol. Biol. Int. 46, 1055–1061 (1998).

    CAS  Google Scholar 

  23. 23.

    Zhang, S., Yuan, J.X.-J., Barrett, K.E. & Dong, H. Role of Na+/Ca2+ exchange in regulating cytosolic Ca2+ in cultured human pulmonary artery smooth muscle cells. Am. J. Physiol. Cell. Physiol. 288, C245-C252 (2005).

    Google Scholar 

  24. 24.

    Zink, D., Sadoni, N. & Stelzer, E. Visualizing chromatin and chromosomes in living cells. Methods 29, 42–50 (2003).

    Article  CAS  Google Scholar 

  25. 25.

    Thévenod, F., Friedmann, J.M., Katsen, A.D. & Hauser, I.A. Up-regulation of multidrug resistance P-glycoprotein via nuclear factor-κB activation protects kidney proximal tubule cells from cadmium- and reactive oxygen species-induced apoptosis. J. Biol. Chem. 275, 1887–1896 (2000).

    Article  Google Scholar 

  26. 26.

    Latinwo, L.M. et al. Effect of cadmium-induced oxidative stress on antioxidative enzymes in mitochondria and cytoplasm of CRL-1439 rat liver cells. Int. J. Mol. Med. 18, 477–481 (2006).

    CAS  Google Scholar 

  27. 27.

    Yang, C.-F., Shen, H.-M., Shen, Y., Zhuang, Z.-X. & Ong, C.-N. Cadmium-induced oxidative cellular damage in human fetal lung fibroblasts (MRC-5 cells). Environ. Health Perspect 105, 712–716 (1997).

    Article  CAS  Google Scholar 

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Correspondence to Seog Woo Rhee.

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Mahto, S.K., Rhee, S.W. A multi-inlet microfluidic device fabricated for in situ detection of multiple cytotoxicity endpoints. BioChip J 6, 48–55 (2012). https://doi.org/10.1007/s13206-012-6107-6

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Keywords

  • Microfluidics
  • Multi-inlet module
  • Cytotoxicity endpoints
  • Multiplex
  • Cytotoxicity assays