Biomedical Microdevices

, Volume 12, Issue 2, pp 253–261 | Cite as

Growth of primary embryo cells in a microculture system

  • Max Villa
  • Sara Pope
  • Joanne Conover
  • Tai-Hsi FanEmail author


We present optimal perfusion conditions for the growth of primary mouse embryonic fibroblasts (mEFs) and mouse embryonic stem cells (mESCs) using a microfluidic perfusion culture system. In an effort to balance nutrient renewal while ensuring the presence of cell secreted factors, we found that the optimal perfusion rate for culturing primary embryonic fibroblasts (mEFs) in our experimental setting is 10 nL/min with an average flow velocity 0.55 μm/s in the microchannel. Primary mEFs may have a greater dependence on cell secreted factors when compared to their immortalized counterpart 3T3 fibroblasts cultured under similar conditions. Both the seeding density and the perfusion rate are critical for the proliferation of primary cells. A week long cultivation of mEFs and mESCs using the microculture system exhibited similar morphology and viability to those grown in a petri dish. Both mEFs and mESCs were analyzed using fluorescence immunoassays to determine their proliferative status and protein expression. Our results demonstrate that a perfusion-based microculture environment is capable of supporting the highly proliferative status of pluripotent embryonic stem cells.


Embryonic stem cells Embryonic fibroblasts Perfusion microculture system 



This work was supported by the State of Connecticut under the Connecticut Stem Cell Research Initiative (Grant 06SCA05). M. Villa and S. Pope thank the UConn-Wesleyan Stem Cell Core for embryonic stem cell culture training.


  1. K.R. Atkuri, L.A. Herzenberg, A.-K. Niemi, T. Cowan, L.A. Herzenberg, Importance of culturing primary lymphocytes at physiological oxygen levels. Proc. Natl. Acad. Sci. 104(11), 4547–4552 (2007)CrossRefGoogle Scholar
  2. S. Avery, K. Inniss, H. Moore, The regulation of self-renewal in human embryonic stem cells. Stem Cells Dev. 15(5), 729–740 (2006)CrossRefGoogle Scholar
  3. S.C. Bendall et al., IGF and FGF cooperatively establish the regulatory stem cell niche of pluripotent human cells in vitro. Nature. 448, 1015–1021 (2007)CrossRefGoogle Scholar
  4. M.R. Bennett et al., Metabolic gene regulation in a dynamically changing environment. Nature. 454, 1119–1122 (2008)CrossRefGoogle Scholar
  5. M.F. Brown, T.P. Gratton, J.A. Stuart, Metabolic rate does not scale with body mass in cultured mammalian cells. Am. J. Physiol. Regul. Integr. Comp. Physiol. 292, R2115–R2121 (2007)Google Scholar
  6. B.G. Chung et al., Human neural stem cell growth and differentiation in a gradient-generating microfluidic device. Lab Chip. 5, 401–406 (2005)CrossRefGoogle Scholar
  7. J.T. Dimos et al., Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science. 321, 1218–1221 (2008)CrossRefGoogle Scholar
  8. L. Edelstein-Keshet, Mathematical models in biology (SIAM, New York, 2005)zbMATHGoogle Scholar
  9. J. El-Ali, P.K. Sorger, K.F. Jensen, Cells on chips. Nature. 442, 403–411 (2006)CrossRefGoogle Scholar
  10. E. Fuchs, T. Tumbar, G. Guasch, Socializing with the neighbors: stem cells and their niches. Cell. 116, 769–778 (2004)CrossRefGoogle Scholar
  11. R. Gómez-Sjöberg, A.A. Leyrat, D.M. Pirone, C.S. Chen, S.R. Quake, Versatile, fully automated, microfluidic cell culture system. Anal. Chem. 79, 8557–8563 (2007)CrossRefGoogle Scholar
  12. L. Hayflick, P.S. Moorhead, The serial cultivation of human diploid cell strains. Exp. Cell. Res. 25, 585–621 (1961)CrossRefGoogle Scholar
  13. C.T. Jordan, Cancer stem cell biology: from leukemia to solid tumors. Curr. Opin. Cell Biol. 16(6), 708–712 (2004)CrossRefGoogle Scholar
  14. L. Kim, Y.-C. Toh, J. Voldman, H. Yu, A practical guide to microfluidic perfusion culture of adherent mammalian cells. Lab Chip. 7, 681–694 (2007)CrossRefGoogle Scholar
  15. L. Kim, M.D. Vahey, H.Y. Lee, J. Voldman, Microfluidic arrays for logarithmically perfused embryonic stem cell culture. Lab Chip. 6, 394–406 (2006)CrossRefGoogle Scholar
  16. N. Korin, A. Bransky, U. Dinnar, S. Levenberg, A parametric study of human fibroblasts culture in a microchannel bioreactor. Lab Chip. 7, 611–617 (2007)CrossRefGoogle Scholar
  17. M.E. Levenstein et al., Basic fibroblast growth factor support of human embryonic stem cell self-renewal. Stem Cells. 24, 568–574 (2006)CrossRefGoogle Scholar
  18. N. Li, A. Tourovskaia, A. Folch, Biology on a chip: microfabrication for studying the behavior of cultured cells. Crit. Rev. Biomed. Eng. 31, 423–488 (2003)CrossRefGoogle Scholar
  19. W.E. Lowry, K. Plath, The many ways to make an iPS cell. Nat. Biotechnol. 26, 1246–1248 (2008)CrossRefGoogle Scholar
  20. J.C. McDonald, G.M. Whitesides, Poly(dimethylsiloxane) as a material for fabricating microfluidic devices. Acc. Chem. Res. 35(7), 491–499 (2002)CrossRefGoogle Scholar
  21. T.C. Merkel, V.I. Bondar, K. Nagai, B.D. Freeman, I. Pinnau, Gas sorption, diffusion, and permeation in poly(dimethylsiloxane). J. Polym. Sci. B Polym. Phys. 38, 415–434 (2000)CrossRefGoogle Scholar
  22. L.J. Millet, M.E. Stewart, J.V. Sweedler, R.G. Nuzzo, M.U. Gillette, Microfluidic devices for culturing primary mammalian neurons at low densities. Lab Chip. 7, 987–994 (2007)CrossRefGoogle Scholar
  23. C.E. Murry, G. Keller, Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell. 132, 661–680 (2008)CrossRefGoogle Scholar
  24. C.P. Ng, M.A. Swartz, Fibroblast alignment under interstitial fluid flow using a novel 3-D tissue culture model. Am. J. Physiol. Heart Circ. Physiol. 284(5), 1171–1777 (2003)Google Scholar
  25. R.L. Panton, Incompressible flow (Wiley, New York, 1996)Google Scholar
  26. I.H. Park et al., Disease-specific induced pluripotent stem cells. Cell. 134, 877–886 (2008)CrossRefGoogle Scholar
  27. R.F. Probstein, Physicochemical hydrodynamics (Wiley, New York, 1994)CrossRefGoogle Scholar
  28. V.K. Ramiya et al., Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells. Nat. Med. 6(3), 278–282 (2000)CrossRefGoogle Scholar
  29. A. Rosenthal, A. Macdonald, J. Voldman, Cell patterning chip for controlling the stem cell microenvironment. Biomaterials. 28(21), 3208–3216 (2007)CrossRefGoogle Scholar
  30. K. Schuster-Gossler et al., Use of coisogenic host blastocysts for efficient establishment of germline chimeras with C57BL/6J ES cell lines. Biotechniques. 31(5), 1022–1026 (2001)Google Scholar
  31. Y. Takagi et al., Dopaminergic neurons generated from monkey embryonic stem cells function in a Parkinson primate model. J. Clin. Invest. 115(1), 102–109 (2005)Google Scholar
  32. K. Takahashi, K. Okita, M. Nakagawa, S. Yamanaka, Induction of pluripotent stem cells from fibroblast cultures. Nature Protocols. 2(12), 3081–3089 (2007a)CrossRefGoogle Scholar
  33. K. Takahashi et al., Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 131, 861–872 (2007b)CrossRefGoogle Scholar
  34. A.M. Taylor et al., A microfluidic culture platform for CNS axonal injury, regeneration and transport. Nature Methods. 2(8), 599–605 (2005)CrossRefGoogle Scholar
  35. J.A. Thomson et al., Embryonic stem cell lines derived from human blastocysts. Science. 282, 1145–1147 (1998)CrossRefGoogle Scholar
  36. G.J. Todaro, H. Green, Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines. J. Cell Biol. 17, 299–313 (1963)CrossRefGoogle Scholar
  37. Y.-C. Toh et al., A novel 3D mammalian cell perfusion-culture system in microfluidic channels. Lab Chip. 7, 302–309 (2007)CrossRefGoogle Scholar
  38. A. Tourovskaia, X. Figueroa-Masot, A. Folch, Long-term microfluidic cultures of myotube microarrays for high-throughput focal stimulation. Nature Protocols. 1(3), 1092–1104 (2006)CrossRefGoogle Scholar
  39. F. Watt, B.L.M. Hogan, Out of Eden: stem cells and their niches. Science. 287, 1427–1430 (2000)CrossRefGoogle Scholar
  40. G.M. Whitesides, The origins and the future of microfluidics. Nature. 44, 368–373 (2006)CrossRefGoogle Scholar
  41. G.M. Whitesides, E. Ostuni, S. Takayama, X. Jiang, D.E. Ingber, Soft lithography in biology and biochemistry. Annu. Rev. Biomed. Eng. 3, 335–373 (2001)CrossRefGoogle Scholar
  42. H. Yu, I. Meyvantsson, I.A. Shkel, D.J. Beebe, Diffusion dependent cell behavior in microenvironments. Lab Chip. 5, 1089–1095 (2005)CrossRefGoogle Scholar
  43. J. Yu et al., Induced pluripotent stem cell lines derived from human somatic cells. Science. 318, 1917–1920 (2007)CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Max Villa
    • 1
  • Sara Pope
    • 2
  • Joanne Conover
    • 2
  • Tai-Hsi Fan
    • 1
    Email author
  1. 1.Department of Mechanical EngineeringUniversity of ConnecticutStorrsUSA
  2. 2.Department of Physiology and NeurobiologyUniversity of ConnecticutStorrsUSA

Personalised recommendations