Biomedical Microdevices

, Volume 11, Issue 6, pp 1155–1165 | Cite as

A microperfused incubator for tissue mimetic 3D cultures

  • Jelena Vukasinovic
  • D. Kacy Cullen
  • Michelle C. LaPlaca
  • Ari Glezer


High density, three-dimensional (3D) cultures present physical similarities to in vivo tissue and are invaluable tools for pre-clinical therapeutic discoveries and development of tissue engineered constructs. Unfortunately, the use of dense cultures is hindered by intra-culture transport limits allowing just a few layer thick cultures for reproducible studies. In order to overcome diffusion limits in intra-culture nutrient and gas availability, a simple scalable microfluidic perfusion platform was developed and validated. A novel perfusion approach maintained laminar flow of nutrients through the culture to meet metabolic need, while removing depleted medium and catabolites. Velocity distributions and 3D flow patterns were measured using microscopic particle image velocimetry. The effectiveness of forced convection laminar perfusion was confirmed by culturing 700 µm thick neural-astrocytic (1:1) constructs at cell density approaching that of the brain (50,000 cells/mm3). At the optimized flow rate of the nutrient medium, the culture viability reached 90% through the full construct thickness at 2 days of perfusion while unperfused controls exhibited widespread cell death. The membrane aerated perfusion platform was integrated within a miniature, imaging accessible enclosure enabling temperature and gas control of the culture environment. Temperature measurements demonstrated fast feedback response to environmental changes resulting in the maintenance of the physiological temperature within 37 ± 0.2°C. Reproducible culturing of tissue equivalents within dynamically controlled environments will provide higher fidelity to in vivo function in an in vitro accessible format for cell-based assays and regenerative medicine.


Incubator Bioreactor Perfusion Convection 3D culture Neuron 


  1. N.H. Bass, H.H. Hess, A. Pope, C. Thalheimer, J. Comp. Neurol. 143, 481 (1971). doi:10.1002/cne.901430405 CrossRefGoogle Scholar
  2. S.N. Bhatia, U.J. Balis, M.L. Yarmush, M. Toner, FASEB J. 13, 1883 (1999)Google Scholar
  3. N.J. Boudreau, P.L. Jones, Biochem. J. 339, 481 (1999). doi:10.1042/0264-6021:3390481 CrossRefGoogle Scholar
  4. K. Brannvall, K. Bergman, U. Wallenquist, S. Svahn, T. Bowden, J. Hilborn, K. Forsberg-Nilsson, J. Neurosci. Res. 85, 2138 (2007). doi:10.1002/jnr.21358 CrossRefGoogle Scholar
  5. V. Braitenberg, J. Comput. Neurosci. 10, 71 (2001). doi:10.1023/A:1008920127052 CrossRefGoogle Scholar
  6. O. Caspi, A. Lesman, Y. Basevitch, A. Gepstein, G. Arbel, I.H. Habib, L. Gepstein, S. Levenberg, Circ. Ress. 100, 263 (2007). doi:10.1161/01.RES.0000257776.05673.ff CrossRefGoogle Scholar
  7. C.S. Chen, M. Mrksich, S. Huang, G.M. Whitesides, D.E. Ingber, Science 276, 1425 (1997). doi:10.1126/science.276.5317.1425 CrossRefGoogle Scholar
  8. Y. Choi, J. Vukasinovic, A. Glezer, M.G. Allen, Biomed. Microdevices. 10, 437 (2008). doi:10.1007/s10544-007-9153-4 CrossRefGoogle Scholar
  9. D.K. Cullen, J. Vukasinovic, A. Glezer, M.C. Laplaca, J. Neural. Eng. 4, 159 (2007). doi:10.1088/1741-2560/4/2/015 CrossRefGoogle Scholar
  10. J.W. Deitmer, Respir. Physiol. 129, 71 (2001). doi:10.1016/S0034-5687(01)00283-3 CrossRefGoogle Scholar
  11. R.P. Dring, J. Fluid. Eng.-T ASME 104, 15 (1982)CrossRefGoogle Scholar
  12. Editorial, Nature 424, 861 (2003) doi: 10.1038/424861b
  13. J. El-Ali, P.K. Sorger, K.F. Jensen, Nature 442, 403 (2006). doi:10.1038/nature05063 CrossRefGoogle Scholar
  14. M.V. Fournier, K.J. Martin, J. Cell Physiol. 209, 625 (2006). doi:10.1002/jcp.20787 CrossRefGoogle Scholar
  15. S. Fox, S. Farr-Jones, L. Sopchak, A. Boggs, H.W. Nicely, R. Khoury, M. Biros, J. Biomol. Screen. 11, 864 (2006). doi:10.1177/1087057106292473 CrossRefGoogle Scholar
  16. P.L. Gabbott, M.G. Stewart, Neuroscience 21, 833 (1987)CrossRefGoogle Scholar
  17. K.O. Hicks, F.B. Pruijn, T.W. Secomb, M.P. Hay, R. Hsu, J.M. Brown, W.A. Denny, M.W. Dewhirst, W.R. Wilson, J. Natl. Cancer. Inst. 98, 1118 (2006). doi:10.1093/jnci/djj306 CrossRefGoogle Scholar
  18. F.P. Incropera, D.P. DeWitt, Fundamentals of heat and mass transfer, 5th edn. (J. Wiley, New York, 2002)Google Scholar
  19. P.A. Kenny, M.J. Bissell, Int. J. Cancer 107, 688 (2003). doi:10.1002/ijc.11491 CrossRefGoogle Scholar
  20. C.J. Kirkpatrick, R.E. Unger, V. Krump-Konvalinkova, K. Peters, H. Schmidt, G. Kamp, J. Mater. Sci. Mater. Med. 14, 677 (2003)CrossRefGoogle Scholar
  21. Y. Kostov, P. Harms, L. Randers-Eichhorn, G. Rao, Biotechnol. Bioeng. 72, 346 (2001). doi:10.1002/1097-0290(20010205)72:3<346::AID-BIT12>3.0.CO;2-X CrossRefGoogle Scholar
  22. S.A. Lelievre, V.M. Weaver, J.A. Nickerson, C.A. Larabell, A. Bhaumik, O.W. Petersen, M.J. Bissell, Proc. Natl. Acad. Sci. USA 95, 14711 (1998). doi:10.1073/pnas.95.25.14711 CrossRefGoogle Scholar
  23. S. Levenberg, J. Rouwkema, M. Macdonald, E.S. Garfein, D.S. Kohane, D.C. Darland, R. Marini, C.A. van Blitterswijk, R.C. Mulligan, P.A. D’Amore, R. Langer, Nat. Biotechnol. 23, 879 (2005). doi:10.1038/nbt1109 CrossRefGoogle Scholar
  24. H. Liu, S.F. Collins, L.J. Suggs, Biomaterials 27, 6004 (2006). doi:10.1016/j.biomaterials.2006.06.016 CrossRefGoogle Scholar
  25. P.J. Magistretti, J. Exp. Biol. 209, 2304 (2006). doi:10.1242/jeb.02208 CrossRefGoogle Scholar
  26. W. Mueller-Klieser, Am. J. Physiol. 273, C1109 (1997)Google Scholar
  27. S.M. Potter, T.B. DeMarse, J. Neurosci. Methods 110, 17 (2001). doi:10.1016/S0165-0270(01)00412-5 CrossRefGoogle Scholar
  28. J. Rouwkema, N.C. Rivron, C.A. van Blitterswijk, Trends Biotechnol. 26, 434 (2008). doi:10.1016/j.tibtech.2008.04.009 CrossRefGoogle Scholar
  29. J.G. Santiago, S.T. Wereley, C.D. Meinhart, D.J. Beebe, R.J. Adrian, Exp. Fluids 25, 316 (1998). doi:10.1007/s003480050235 CrossRefGoogle Scholar
  30. M. Schindler, E.K.A. Nur, I. Ahmed, J. Kamal, H.Y. Liu, N. Amor, A.S. Ponery, D.P. Crockett, T.H. Grafe, H.Y. Chung, T. Weik, E. Jones, S. Meiners, Cell Biochem. Biophys. 45, 215 (2006). doi:10.1385/CBB:45:2:215 CrossRefGoogle Scholar
  31. K.S. Smalley, M. Lioni, M. Herlyn, In Vitro Cell Dev. Biol. Anim. 42, 242 (2006). doi:10.1290/0604027.1 CrossRefGoogle Scholar
  32. L. Stoppini, P.A. Buchs, D. Muller, J. Neurosci. Methods 37, 173 (1991)CrossRefGoogle Scholar
  33. F. Sultan, V. Braitenberg, J. Hirnforsch. 34, 79 (1993)Google Scholar
  34. M.A. Swartz, M.E. Fleury, Annu. Rev. Biomed. Eng. 9, 229 (2007). doi:10.1146/annurev.bioeng.9.060906.151850 CrossRefGoogle Scholar
  35. C.H. Thomas, J.H. Collier, C.S. Sfeir, K.E. Healy, Proc. Natl. Acad. Sci. USA 99, 1972 (2002). doi:10.1073/pnas.032668799 CrossRefGoogle Scholar
  36. M. Tsacopoulos, P.J. Magistretti, J. Neurosci. 16, 877 (1996)Google Scholar
  37. J. Voldman, M.L. Gray, M.A. Schmidt, Annu. Rev. Biomed. Eng. 1, 401 (1999). doi:10.1146/annurev.bioeng.1.1.401 CrossRefGoogle Scholar
  38. D. Walpita, E. Hay, Nat. Rev. Mol. Cell. Biol. 3(2), 137 (2002). doi:10.1038/nrm727 CrossRefGoogle Scholar
  39. D. Wang, W. Liu, B. Han, R. Xu, Curr. Pharm. Biotechnol. 6, 397 (2005)CrossRefGoogle Scholar
  40. V.M. Weaver, O.W. Petersen, F. Wang, C.A. Larabell, P. Briand, C. Damsky, M.J. Bissell, J. Cell Biol. 137, 231 (1997)CrossRefGoogle Scholar
  41. R.W. Williams, K. Herrup, Annu. Rev. Neurosci. 11, 423 (1988). doi:10.1146/ CrossRefGoogle Scholar
  42. M.H. Wu, J.P. Urban, Z.F. Cui, Z. Cui, X. Xu, Biotechnol. Prog. 23, 430 (2007). doi:10.1021/bp060024v CrossRefGoogle Scholar
  43. C. Yamamoto, H. McIlwain, J. Neurochem. 13, 1333 (1966). doi:10.1111/j.1471-4159.1966.tb04296.x CrossRefGoogle Scholar
  44. M.H. Zaman, L.M. Trapani, A.L. Sieminski, D. Mackellar, H. Gong, R.D. Kamm, A. Wells, D.A. Lauffenburger, P. Matsudaira, Proc. Natl. Acad. Sci. U.S.A. 103, 10889 (2006). doi:10.1073/pnas.0604460103 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Jelena Vukasinovic
    • 1
  • D. Kacy Cullen
    • 2
  • Michelle C. LaPlaca
    • 2
  • Ari Glezer
    • 1
  1. 1.Woodruff School of Mechanical EngineeringGeorgia Institute of TechnologyAtlantaUSA
  2. 2.Coulter Department of Biomedical EngineeringGeorgia Institute of TechnologyAtlantaUSA

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