Biomedical Microdevices

, Volume 14, Issue 2, pp 313–323

Micro-ring structures stabilize microdroplets to enable long term spheroid culture in 384 hanging drop array plates

  • Amy Y. Hsiao
  • Yi-Chung Tung
  • Chuan-Hsien Kuo
  • Bobak Mosadegh
  • Rachel Bedenis
  • Kenneth J. Pienta
  • Shuichi Takayama


Using stereolithography, 20 different structural variations comprised of millimeter diameter holes surrounded by trenches, plateaus, or micro-ring structures were prepared and tested for their ability to stably hold arrays of microliter sized droplets within the structures over an extended period of time. The micro-ring structures were the most effective in stabilizing droplets against mechanical and chemical perturbations. After confirming the importance of micro-ring structures using rapid prototyping, we developed an injection molding tool for mass production of polystyrene 3D cell culture plates with an array of 384 such micro-ring surrounded through-hole structures. These newly designed and injection molded polystyrene 384 hanging drop array plates with micro-rings were stable and robust against mechanical perturbations as well as surface fouling-facilitated droplet spreading making them capable of long term cell spheroid culture of up to 22 days within the droplet array. This is a significant improvement over previously reported 384 hanging drop array plates which are susceptible to small mechanical shocks and could not reliably maintain hanging drops for longer than a few days. With enhanced droplet stability, the hanging drop array plates with micro-ring structures provide better platforms and open up new opportunities for high-throughput preparation of microscale 3D cell constructs for drug screening and cell analysis.


Micro-ring Spheroid High-throughput 3D culture Hanging drop Stereolithography 


  1. D. Bakhos, S. Velut, A. Robier, M. Al zahrani, E. Lescanne, Otol. Neurotol. 31, 328–334 (2010)CrossRefGoogle Scholar
  2. V. Chan, P. Zorlutuna, J.H. Jeong, H. Kong, R. Bashir, Lab. Chip. 10, 2062–2070 (2010)CrossRefGoogle Scholar
  3. M.N. Cooke, J.P. Fisher, D. Dean, C. Rimnac, A.G. Mikos, J. Biomed, Mater. Res. B Appl. Biomater. 64, 65–69 (2003)CrossRefGoogle Scholar
  4. B. Fallahi, M. Foroutan, S. Motayalli, M. Dujoyny, S. Limaye, Neurol. Res. 21, 281–286 (1999)Google Scholar
  5. M. Foroutan, B. Fallahi, S. Mottayalli, M. Dujoyny, Crit. Rev. Neurosurg. 8, 203–208 (1998)CrossRefGoogle Scholar
  6. J. Friedrich, C. Seidel, R. Ebner, L.A. Kunz-Schughart, Nat. Protoc. 4, 309–324 (2009)CrossRefGoogle Scholar
  7. A.A. Gill, F. Claevssens, Methods Mol. Biol. 695, 309–321 (2011)CrossRefGoogle Scholar
  8. F. Hirschhaeuser, H. Menne, C. Dittfeld, J. West, W. Mueller-Klieser, L.A. Kunz-Schughart, J. Biotechnol. 148, 3–15 (2010)CrossRefGoogle Scholar
  9. L.M. Kalikin, A. Schneider, M.A. Thakur, Y. Fridman, L.B. Griffin, R.L. Dunn, T.J. Rosol, R.B. Shah, A. Rehemtulla, L.K. McCauley, K.J. Pienta, Cancer Biol. Ther. 2, 656–660 (2003)Google Scholar
  10. Y. Kalinin, V. Berejnov, R.E. Thorne, Microfluid. Nanofluidics 5, 449–454 (2008)CrossRefGoogle Scholar
  11. S. Kawata, H.B. Sun, T. Tanaka, K. Takada, Nature 412, 697–698 (2001)CrossRefGoogle Scholar
  12. J.M. Kelm, M. Fussenegger, Trends Biotechnol. 22, 195–202 (2004)CrossRefGoogle Scholar
  13. M. Kettner, P. Schmidt, S. Potente, F. Ramsthaler, M. Schrodt, J. Forensic. Sci. 56, 1015–1017 (2011)CrossRefGoogle Scholar
  14. A. Khademhosseini, R. Langer, J. Borenstein, J.P. Vacanti, Proc. Natl. Acad. Sci. USA 103, 2480–2487 (2006)CrossRefGoogle Scholar
  15. K. Kobayashi, K. Ikuta, IEEE/ASME Intern. Conf. Adv. Intell. Mechatron. 1–3, 661–666 (2007)Google Scholar
  16. K. Kobayashi, K. Ikuta, Appl. Phys. Lett. 92, 262505 (2008)CrossRefGoogle Scholar
  17. L.A. Kunz-Schughart, J.P. Freyer, F. Hofstaedter, R. Ebner, J. Biomol, Screen 9, 273–285 (2004)Google Scholar
  18. J.W. Lee, P.X. Lan, B. Kim, G. Lim, D.W. Cho, J. Biomed, Mater. Res. B Appl. Biomater. 87, 1–9 (2008)CrossRefGoogle Scholar
  19. W.G. Lee, D. Ortmann, M.J. Hancock, H. Bae, A. Khademhosseini, Tissue Eng. Part C Methods 16, 249–259 (2010)CrossRefGoogle Scholar
  20. S.J. Leigh, C.P. Purssell, J. Bowen, D.A. Hutchins, J.A. Covington, D.R. Billson, Sens. Actuator A-Phys. 168, 66–71 (2011)CrossRefGoogle Scholar
  21. T.W. Lim, Y. Son, Y.J. Jeong, D.Y. Yang, H.J. Kong, K.S. Lee, D.P. Kim, Lab. Chip. 11, 100–103 (2011)CrossRefGoogle Scholar
  22. R.Z. Lin, H.Y. Chang, Biotechnol. J. 3, 1172–1184 (2008)MathSciNetCrossRefGoogle Scholar
  23. H. Lopponen, T. Holma, M. Sorri, L. Jyrkinen, V. Karhula, A. Kojyula, E. Ilkko, J. Laitinen, J. Kojyukangas, J. Oikarinen, O. Alamaki, Acta Otolaryngol. Suppl. 529, 47–49 (1997)CrossRefGoogle Scholar
  24. G. Mapili, Y. Lu, S. Chen, K. Roy, J. Biomed, Mater. Res. B. Appl. Biomater. 75, 414–424 (2005)CrossRefGoogle Scholar
  25. S. Maruo, K. Ikuta, Sens. Actuator A-Phys. 100, 70–76 (2002)CrossRefGoogle Scholar
  26. S. Maruo, H. Inonue, Appl. Phys. Lett. 89, 144101 (2006)CrossRefGoogle Scholar
  27. S. Maruo, K. Ikuta, H. Korogi, J. Microelectromech, Syst. 12, 533–539 (2003)Google Scholar
  28. S. Maruo, A. Takaura, Y. Saito, Opt. Express 17, 18525–18532 (2009)CrossRefGoogle Scholar
  29. F.P.W. Melchels, K. Bertoldi, R. Gabbrielli, A.H. Velders, J. Feijen, D.W. Grijpma, Biomaterials 27, 6909–6916 (2010a)CrossRefGoogle Scholar
  30. F.P.W. Melchels, J. Feijen, D.W. Grijpma, Biomaterials 31, 6121–6130 (2010b)CrossRefGoogle Scholar
  31. W. Mueller-Klieser, Am. J. Physiol. 273, C1109–C1123 (1997)Google Scholar
  32. R.J. Narayan, A. Doraiswamy, D.B. Chrisey, B.N. Chichkov, Mater. Today 13, 42–48 (2010)CrossRefGoogle Scholar
  33. W.S. Paiva, R. Amorim, D.A. Bezerra, M. Masini, Arg. Neuropsiquiatr. 65, 443–445 (2007)CrossRefGoogle Scholar
  34. S.H. Park, D.Y. Yang, K.S. Lee, Laser Photon. Rev. 3, 1–11 (2009)CrossRefGoogle Scholar
  35. K. Rudman, C. Hoekzema, J. Rhee, Facial Plast. Surg. 27, 358–365 (2011)CrossRefGoogle Scholar
  36. H. Selvaraj, B. Tan, K. Venkatakrishnan, J. Micromech. Microeng. 21, 075018 (2011)CrossRefGoogle Scholar
  37. J.H. Shin, J.W. Lee, J.H. Jung, D.W. Cho, G. Lim, J. Mater, Sci. 46, 5282–5287 (2011)Google Scholar
  38. Y.C. Tung, A.Y. Hsiao, S.G. Allen, Y.S. Torisawa, M. Ho, S. Takayama, Analyst 136, 473–478 (2011)CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Amy Y. Hsiao
    • 1
  • Yi-Chung Tung
    • 1
    • 2
  • Chuan-Hsien Kuo
    • 1
  • Bobak Mosadegh
    • 1
  • Rachel Bedenis
    • 3
  • Kenneth J. Pienta
    • 3
    • 4
  • Shuichi Takayama
    • 1
    • 5
    • 6
  1. 1.Department of Biomedical EngineeringUniversity of MichiganAnn ArborUSA
  2. 2.Research Center for Applied SciencesAcademia SinicaTaipeiTaiwan
  3. 3.Department of Internal MedicineUniversity of Michigan Medical SchoolAnn ArborUSA
  4. 4.Department of UrologyUniversity of Michigan Medical SchoolAnn ArborUSA
  5. 5.Macro Molecular Science and EngineeringUniversity of MichiganAnn ArborUSA
  6. 6.School of Nano-Biotechnology and Chemical Engineering WCU ProjectUNISTUlsanRepublic of Korea

Personalised recommendations