Biomedical Microdevices

, Volume 6, Issue 4, pp 325–339 | Cite as

NanoLiterBioReactor: Long-Term Mammalian Cell Culture at Nanofabricated Scale

  • Ales Prokop
  • Zdenka Prokop
  • David Schaffer
  • Eugene Kozlov
  • John Wikswo
  • David Cliffel
  • Franz Baudenbacher

Abstract

There is a need for microminiaturized cell-culture environments, i.e. NanoLiter BioReactors (NBRs), for growing and maintaining populations of up to several hundred cultured mammalian cells in volumes three orders of magnitude smaller than those contained in standard multi-well screening plates. These devices would enable the development of a new class of miniature, automated cell-based bioanalysis arrays for monitoring the immediate environment of multiple cell lines and assessing the effects of drug or toxin exposure.

We fabricated NBR prototypes, each of which incorporates a culture chamber, inlet and outlet ports, and connecting microfluidic conduits. The fluidic components were molded in polydimethylsiloxane (PDMS) using soft-lithography techniques, and sealed via plasma activation against a glass slide, which served as the primary culture substrate in the NBR. The input and outlet ports were punched into the PDMS block, and enabled the supply and withdrawal of culture medium into/from the culture chamber (10–100 nL volume), as well as cell seeding. Because of the intrinsically high oxygen permeability of the PDMS material, no additional CO2/air supply was necessary.

The developmental process for the NBR typically employed several iterations of the following steps: Conceptual design, mask generation, photolithography, soft lithography, and proof-of-concept culture assay. We have arrived at several intermediate designs. One is termed “circular NBR with a central post (CP-NBR),” another, “perfusion (grid) NBR (PG-NBR),” and a third version, “multitrap (cage) NBR (MT-NBR),” the last two providing total cell retention.

Three cells lines were tested in detail: a fibroblast cell line, CHO cells, and hepatocytes. Prior to the culturing trials, extensive biocompatibility tests were performed on all materials to be employed in the NBR design. To delineate the effect of cell seeding density on cell viability and survival, we conducted separate plating experiments using standard culture protocols in well-plate dishes. In both experiments, PicoGreen assays were used to evaluate the extent of cell growth achieved in 1–5 days following the seeding. Low seeding densities resulted in the absence of cell proliferation for some cell lines because of the deficiency of cell-cell and extracellular matrix (ECM)-cell contacts. High viabilities were achieved in all designs.

We conclude that an instrumented microfluidics-based NanoBioReactor (NBR) will represent a dramatic departure from the standard culture environment. The employment of NBRs for mammalian cell culture opens a new paradigm of cell biology, so far largely neglected in the literature.

nanobioreactor long-term mammalian culture 

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References

  1. J.W. Allen and S.N. Bhatia, Formation of steady-state oxygen gradients in vitro. Biotechnol. Bioeng. 82, 253 (2003).Google Scholar
  2. R.R. Balcarcel and L.M. Clark, Metabolic screening of mammalian cell cultures using well-plates. Biotechnol. Prog. 19,98 (2003).Google Scholar
  3. H. Becker and C. Gartner, Polymer based micro-reactors. Revs. Molec. Biotechnol. 82, 89 (2001).Google Scholar
  4. J.T. Borenstein, H. Terai, K.R. King, E.J. Weinberg, M.R. Kaazempur-Mofrad, and J.P. Vacanti, Microfabrication technology for vascularized tissue engineering. Biomed. Microdev. 4, 167 (2002).Google Scholar
  5. C.T. Bratten, P.H. Cobbold, and J.M. Cooper, Micromachining sensors for electrochemical measurement in subnanoliter volumes. Analyt. Chem. 69, 253 (1997).Google Scholar
  6. M. Brischwein, E.R. Motrescu, E. Cabala, A.M. Otto, H. Grothe, and B. Wolf, Functional cellular assays with multiparametric silicon sensor. Lab. Chip. 3, 234 (2003).Google Scholar
  7. S.C. Cahrati and S.A. Stern, Diffusion of gases in silicone polymers: Molecular dynamics simulations. Macromol. 31, 5529 (1998).Google Scholar
  8. W.-J. Chang, D. Akin, M. Sedlak, M.R. Ladisch, and R. Bashir, Poly(dimethylsiloxane) (PDMS) and silicon hybrid biochip for bac-terial culture. Biomed. Microdev. 6, 281 (2003).Google Scholar
  9. J.M. Cooper, Towards electronic Petri dishes and picolitre-scale single-cell techniques. Trends Biotechnol. 17, 226 (1999).Google Scholar
  10. E.J. Crosby, Experiments in Transport Phenomena (Wiley, New York, 1961).Google Scholar
  11. I. De Bo, H. Van Langenhove, P. Pruuost, J. De Neve, J. Pieters, I.F.J. Vakelecom, and E. Dick, Investigation of the permeability and selec-tivity of gases and volatile organic compounds for polydimethylsilox-ane membranes. J. Membrane. Sci. 215, 303 (2003).Google Scholar
  12. K. Efimenko, W.E. Wallace, and J. Genzer, Surface modification of Sylgard-184 Poly(dimethyl siloxane) networks by ultraviolet and ul-traviolet/ ozone treatment. J. Coll. Interface Sci. 254, 306 (2002).Google Scholar
  13. S.E. Eklund, D. Taylor, E. Kozlov, A. Prokop, and D.E. Cliffel, A micro-physiometer for simultaneous measurement of changes in extracellu-lar glucose, lactate, oxygen, and acidification rate. Anal. Chem. 76, 519 (2004).Google Scholar
  14. A. Folch and M. Toner, Microengineering of cellular interactions. Ann Rev Biomed Eng 2, 227 (2000).Google Scholar
  15. M. Gerritsen, A. Kros, V. Sprakel, L.A. Lutterman, R.J.M. Nolte, and J.A. Jansen, Biocompatibility evaluation of sol-gel coatings for sub-cutaneously implantable glucose sensors. Biomat. 21, 71 (2000).Google Scholar
  16. A. Ghanem and M.L. Shuler, Characterization of a perfusion reactor utilizing mammalian cells on microcarrier beads. Biotechnol. Progr. 16, 471 (2000).Google Scholar
  17. A. Grodrian, J. Metze, Th. Henkel, M. Roth, and J.M. Kohler, Segmented flowgeneration by chip reactors for highly parallelized cell cultivation. In Biomedical Applications of Micro-and Nanoengineering, edited by D.V. Nicolau and A.P. Lee, Proc. SPIE 4937, 174 (2002).Google Scholar
  18. F. Hafner, Cytosensor microphysiometer: Technology and recent appli-cations. Biosensors Bioelectr. 15, 149 (2000).Google Scholar
  19. S. Hediger, A. Sayah, J.D. Horisberger, and M.A.M. Hijs, Modular mi-crosystem for epithelial cell culture and electrical characterization. Biosens. Bioelectr. 16, 689 (2001).Google Scholar
  20. W.-H. Huang, W. Chang, Z. Zhang, D.-W. Pang, Z.-L. Wang, J.-K. Cheng, and D.-F. Cui, Transport, location, and quantal release mon-itoring of single cells on a microfluidic device. Anal. Chem. 76, 483 (2004).Google Scholar
  21. E.W.H. Jager, C. Immerstrand, K.H. Peterson, K.-E. Magnusson, I. Lundstrom, and O. Inganas, The cell clinic: Closable microvials for single cell studies. Biomed Microdev 4, 177 (2002).Google Scholar
  22. E.A. Johannessen, J.M. Weaver, L. Bourova, P. Svoboda, P.H. Gobbold, and J.M. Cooper, Micromachined nanocalorimetric sen-sor for ultra-low-volume cell-based assays. Analyt. Chem. 74, 2190 (2002).Google Scholar
  23. D.R. Jung, R. Kapur, T. Adams, K.A. Giuliano, M. Mrksich, H.G. Craighead, and D.L. Taylor, Topographical and physicochemical mod-ification of material surface to enable patterning of living cells. Crit. Rev. Biotechnol. 21, 111 (2001).Google Scholar
  24. J. Kim, M.K. Chaudhury, and M.J. Owen, Hydrophobic recovery of poly-dimethylsiloxane elastomer exposed to partial electrical discharge. J. Coll. Interface. Sci. 226, 231 (2000).Google Scholar
  25. M.W. Konrad, B. Storrie, D.A. Glaser, and L.H. Thompson, Clonal vari-ation in colony morphology and growth of CHOcells cultured on agar. Cell 10, 305 (1977).Google Scholar
  26. E. Leclerc, Y. Sakai, and T. Fujii, Cell culture in 3-dimensional microflu-idic structure of PDMS (polydimethylsiloxane). Biomed. Microdev. 5, 109 (2002).Google Scholar
  27. E. Leclerc, Y. Sakai, and T. Fujii, Microfluidic PDMS (Polydimethyl-siloxane) bioreactor for large-scale culture of hepatocytes. Biotechnol. Progr. 20, 750 (2004).Google Scholar
  28. S.J. Lee and S.Y. Lee, Micro total analysis system (μ-TAS) in biotech-nology. Appl. Microbiol. Biotechnol. 64, 289 (2004).Google Scholar
  29. M.M. Maharbiz, W.J. Holtz, R.T. Howe, and J.D. Keasling, Microbiore-actor arrays with parametric control for high-throughput experimen-tation. Biotechnol. Bioeng. 85, 376 (2004).Google Scholar
  30. A. Manz, N. Graber, and H.M. Widmer, Miniaturized total chemical analysis systems: A novel concept for chemical screening. Sensors Act B1, 244 (1990).Google Scholar
  31. J.C. McDonald and G.M. Whitesides, Poly(dimethylsiloxane) as a mate-rial for fabricating microfluidic devices. Account Chem. Res. 35, 491 (2002).Google Scholar
  32. T.C. Mekel, V.I. Bondar, K. Nagai, B.D. Freeman, and I. Pinnau, Gas sorption, diffusion, and permeation in poly(dimethylsiloxane). J. Poly-mer. Sci., Polymer. Phys. B 38, 415 (2000).Google Scholar
  33. G. Michalopoulos, H.D. Cianciulli, A.R. Novotny, A.D. Kligerman, S.C. Strom, and R.L. Jirtle, Liver regeneration studies with rat hepatocytes in primary culture. Cancer Res 42, 4673 (1982).Google Scholar
  34. M. Moisan, J. Barbeau, S. Moreau, J. Pelletier, M. Tabrizian, and L.H. Yahia, Low-temperature sterilization using gas plasmas: A review of the experiments and an analysis of the inactivation mechanisms. Int. J. Phamaceut. 226, 1 (2001).Google Scholar
  35. F. Moussy, D.J. Harrison, and R.V. Rajotte, Aminiaturized Nafion-based glucose sensor: In vitro and in vivo evaluation in dogs. Int. J. Artif. Organs. 17, 88 (1994).Google Scholar
  36. A.B. Newman, Trans. Amer. Inst. Chem. Eng. 27, 203 (1931).Google Scholar
  37. J.Y. Oldshue, Fluid Mixing Technology (McGraw-Hill, NewYork, 1983).Google Scholar
  38. C.H. Park, B.F. Kimler, and T.K. Smith, Clonogenic assay combined with flow cytometric cell sorting for cell-cycle analysis of human leukemic colony-forming cells. Anticancer. Res. 7, 129 (1987).Google Scholar
  39. P.A. Parsons-Wingerter and W.M. Saltzman, Growth versus function in the three-dimensional culture of single and aggregated hepatocytes within collagen gels. Biotechnol. Progr. 9, 600 (1993).Google Scholar
  40. J. Pomp, J.L. Wike, I.J.M. Ouwerkerk, C. Hoogstraten, J. Dvelaar, P.I. Schrier, J.W.H. Leer, H.D. Thames, and W.A. Brock, Cell density de-pendent plating efficiency affects outcome and interpretation of colony forming assays. Radiother. Oncol. 40, 121 (1996).Google Scholar
  41. M.J. Powers, K. Domansky, M.R. Kaazempur-Mofrad, A. Kalezi, A. Capitano, A. Upadhyaya, P. Kurzawski, K.E. Wack, D.B. Stolz, R. Kamm, and L.G. Griffith, A microfabricated array bioreactor for per-fused 3D liver culture. Biotechnol. Bioeng. 78, 257 (2002).Google Scholar
  42. A. Prokop and R.K. Bajpai, The sensitivity of biocatalysts to hydro-dynamic shear stress. In Advances in Applied Microbiology, edited by A. Laskin (Academic Press, New York, 1992), vol. 37, pp. 165–232.Google Scholar
  43. A. Prokop, Systems analysis and synthesis in biology and biotechnology. Int. J. Gen. Syst. 8, 1 (1982).Google Scholar
  44. A. Prokop, Challenges in commercial biotechnology. Part I: Product, process and market discovery. Advan. Appl. Microbiol. 40, 95 (1995).Google Scholar
  45. A. Sin, K.C. Chin, M.H. Jamil, Y. Kostov, G. Rao, and M.L. Shuller, The design and fabrication of three-chamber microscale cell culture devices with integrated dissolved oxygen sensors. Biotechnol. Progr. 20, 338 (2004).Google Scholar
  46. K. Slater, Cytotoxicity tests for high-throughput drug discovery. Curr. Opin. Biotechnol. 12, 70 (2001).Google Scholar
  47. J.G. Steele, G. Johnson, W.D. Norris, and P.A. Underwood, Adhesion and growth of cultured human endothelial cells on perfluorosulphonate: Role of vitronectin and fibronectin in cell attachment. Biomat. 12, 531 (1991).Google Scholar
  48. W.R. Tolbert, Perfusion culture systems for production of mammalian cell biomolecules. In Large Scale Mammalian Culture, edited by J. Feder and W.R. Tolbert (Academic Press, New York, 1985), pp. 97–123.Google Scholar
  49. T.G. van Kooten, J.F. Whitesides, and A.F. von Recum, Influence of silicone (PDMS) surface texture on human skin fibroblast proliferation as determined by cell cycle analysis. J. Biomed. Mater. Res. (Appl. Biomater.) 43, 1 (1998).Google Scholar
  50. K. Viravaidya and M.L. Shuller, Incorporation of 3T3-L1 cells to mimic bioaccumulation in a microscale cell culture analog device for toxicity studies. Biotechnol. Progr. 20, 590 (2004).Google Scholar
  51. J. Voldman, M.L. Gray, and M.A. Schmidt, Microfabrication in biology and medicine. Annu. Rev. Biomed. Eng. 1, 401 (1999).Google Scholar
  52. B. Wang, Z. Abdulali-Kanji, E. Dodwell, J.H. Horton, and R.D. Oleschuk, Surface characterization using chemical force microscopy and the flow performance of modified polydimethylsiloxane for mi-crofluidic device applications. Eletrophoresis 24, 1442 (2003).Google Scholar
  53. K.F. Weibezahn, G. Knedlitschek, H. Dertinger, W. Bier, Th. Schiller, and K. Schubert, Reconstruction of tissue layers in mechanically pro-cessed microstructures. J. Exp. Clin. Cancer. Res. 14(Suppl. 1), S41 (1995).Google Scholar
  54. G.M. Whitesides, E. Ostuni, S. Takayama, X. Jiang, and D.E. Ingber. Soft lithography in biology and biochemistry. Annu. Rev. Biomed. Engr. 3, 335 (2001).Google Scholar
  55. A.R. Wheeler, W.R. Throndset, R.J. Whelan, A.M. Leach, R.N. Zare, Y.-H. Liao, K. Farrell, I.D. Manger, and A. Daridon, Microfluidic device for single-cell analysis. Anal Chem 75, 3249 (2003).Google Scholar
  56. Y. Yang and R.R. Balcarcel, Determination of carbon dioxide production rates for mammalian cells in 24-well plates. Bio. Techniques. 36, 286 (2004).Google Scholar
  57. A. Zanzotto, N. Szita, P. Boccazzi, P. Lessard, A.J. Sinskey, and K.F. Jensen, Membrane-aerated microbioreactor for high-throughput bio-processing. Biotechnol. Bioeng. 87, 243 (2004).Google Scholar
  58. T.J. Zieziulewicz, D.W. Unfricht, N. Hadjout, M.A. Lynes, and D.A. Lawrence, Shrinking the biologic world—Nanobiotechnologies for toxicology. Toxicol. Sci. 74, 235 (2003).Google Scholar

Copyright information

© Kluwer Academic Publishers 2004

Authors and Affiliations

  • Ales Prokop
    • 1
    • 2
  • Zdenka Prokop
    • 1
  • David Schaffer
    • 3
  • Eugene Kozlov
    • 2
  • John Wikswo
    • 1
  • David Cliffel
    • 1
  • Franz Baudenbacher
    • 4
  1. 1.NanoDelivery, Inc.Nashville
  2. 2.Chemical EngineeringVanderbilt UniversityNashville
  3. 3.Mechanical EngineeringUSA
  4. 4.Biomedical EngineeringUSA

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