Skip to main content
SpringerLink
Log in

Efficient Formation of Three Dimensional Spheroids of Primary Hepatocytes Using Micropatterned Multi-Well Plates

  • Article
  • Published:
Macromolecular Research Aims and scope Submit manuscript

Abstract

Primary hepatocytes constitute 60-80% of liver where they play a vital role in metabolism, synthesis and storage such as vitamins, carbohydrates, proteins. When hepatocytes are grown in vitro as monolayers, they rapidly lose their phenotypic characteristics and functional abilities. We successfully developed a threedimensional (3D) culturing system for rat primary hepatocytes using micropatterned multi-well plates. The efficiency, morphology, cell viability and functionality (albumin secretion and urea synthesis) of hepatocyte spheroids produced by 3D and monolayer culture were compared. In monolayer culture, hepatocytes did not maintain an intact morphology and their viabilities started to decline after 4 days. On the other hand, hepatocyte spheroids were prepared with various numbers of single hepatocytes. It was confirmed that 150 hepatocytes per spheroid were optimum with incorporation efficiencies of around 95% as well as obtained spheroids were more homogenous in shape and size. Additionally, the secretion of albumin and production of urea by these spheroids were significantly greater than those of cells in monolayer culture. Our results show the described method could potentially be used in liver tissue regeneration, new drug discovery or toxicology studies.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Y. Umehara, K. Hakamada, K. Seino, K. Aoki, Y. Toyoki, and M. Sasaki, Surgery, 130, 513 (2001).

    CAS  PubMed  Google Scholar 

  2. R. F. Meirelles Junior, P. Salvalaggio, M. B. Rezende, A. S. Evangelista, B. D. Guardia, C. E., Matielo, D. B. Neves, F. L. Pandullo, G. E. Felga, J. A. Alves, L. A. Curvelo, L. G. Diaz, M. B. Rusi, M. Viveiros Mde, M. D. Almeida, P. T. Pedroso, R. A. Rocco, and S. P. Meira Filho, Einstein (Sao Paulo, Brazil), 13, 149 (2015).

    Google Scholar 

  3. C. M. Smith, D. B. Davies, and M. A. McBride, Clin. Transpl., 23 (1999).

    Google Scholar 

  4. A. A. Demetriou, J. Whiting, S. M. Levenson, N. R. Chowdhury, R. Schechner, S. Michalski, D. Feldman, and J. R. Chowdhury, Ann. Surg., 204, 259 (1986).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. I. J. Fox and J. Roy-Chowdhury, J. Hepatol., 40, 878 (2004).

    CAS  PubMed  Google Scholar 

  6. H. Nagata, M. Ito, J. Cai, A. S. Edge, J. L. Platt, and I. J. Fox, Gastroenterology, 124, 422 (2003).

    PubMed  Google Scholar 

  7. M. Rojkind, Z. Gatmaitan, S. Mackensen, M. A. Giambrone, P. Ponce, and L. M. Reid, J. Cell Biol., 87, 255 (1980).

    CAS  PubMed  Google Scholar 

  8. M. Shri, H. Agrawal, P. Rani, D. Singh, and S. K. Onteru, Sci. Rep., 7, 1203 (2017).

    PubMed  PubMed Central  Google Scholar 

  9. J. M. Cha, H. Park, E. K. Shin, J. H. Sung, O. Kim, W. Jung, O. Y. Bang, and J. Kim, Biofabrication, 9, 035006 (2017).

    PubMed  Google Scholar 

  10. R. S. Teotia, D. Katila, A. K. Singh, S. K. Verma, S. S. Kadam, and J. R. Bellare, ACS Biomater. Sci. Eng., 1, 6 (2015).

    Google Scholar 

  11. H. L. Jiang, Y. K. Kim, K. H, Cho, Y. C. Jang, Y. J. Choi, J. H., Chung, and C. S. Cho, Int. J. Stem Cells, 3, 69 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. J. W. Haycock, Methods Mol. Biol. (Clifton, N.J.), 695, 1 (2011).

    CAS  Google Scholar 

  13. J. M. Cha, H. Bae, N. Sadr, S. Manoucheri, F. Edalat, K. Kim, S. B. Kim, I. K. Kwon, Y. S. Hwang, and A. Khademhosseini, Macromol. Res., 23, 245 (2015).

    CAS  Google Scholar 

  14. L. Schukur, P. Zorlutuna, J. M. Cha, H. Bae, and A. Khademhosseini, Adv. Healthc. Mater., 2, 195 (2013).

    CAS  PubMed  Google Scholar 

  15. J. Y. Ko and G. I. Im, Biotechnol. Lett., 38, 1967 (2016).

    CAS  PubMed  Google Scholar 

  16. Y. Y. Choi, B. G. Chung, D. H. Lee, A. Khademhosseini, J. H. Kim, and S. H. Lee, Biomaterials, 31, 4296 (2010).

    CAS  PubMed  Google Scholar 

  17. A. Kang, H. I. Seo, B. G. Chung, and S. H. Lee, Nanomedicine, 11, 1153 (2015).

    CAS  PubMed  Google Scholar 

  18. M. Théry, J. Cell. Sci., 123, 24 (2010).

    Google Scholar 

  19. H. Otsuka, A. Hirano, Y. Nagasaki, T. Okano, Y. Horiike, and K. Kataoka, Chem. Bio. Chem., 5, 6 (2004).

    Google Scholar 

  20. S. M. L. Lee, C. Schelcher, M. Demmel, M. Hauner, and W. E. Thasler, J. Vis. Exp., 79, 50615 (2013).

    Google Scholar 

  21. L. Shen, A. Hillebrand, D. Q. Wang, and M. Liu, J. Vis. Exp., 64, 3917 (2012).

    Google Scholar 

  22. H. L. Bank, In Vitro Cell Dev. Biol., 24, 266 (1988).

    CAS  PubMed  Google Scholar 

  23. T. T. Pham,T. T. Nguyen, S. Pathak, S. Regmi, H. T., Nguyen, T. H. Tran, C. S. Yong, J. O. Kim, P. H. Park, M. H. Park, Y. K. Bae, J. U. Choi, Y. Byun, C. H. Ahn, S. Yook, and J. H. Jeong, Biomaterials, 154, 182 (2018).

    CAS  PubMed  Google Scholar 

  24. N. Ismail, M. U. Imam, N. H. Azmi, S. F., Fathy, J. B. Foo, and M. F. Abu Bakar, BMC Complement. Altern. Med., 14, 467 (2014).

    PubMed  PubMed Central  Google Scholar 

  25. J. C. Dunn, M. L. Yarmush, H. G. Koebe, and R. G. Tompkins, FASEB J., 3, 174 (1989).

    CAS  PubMed  Google Scholar 

  26. S. Regmi and J. H. Jeong, Macromol. Res., 24, 1037 (2016).

    CAS  Google Scholar 

  27. E. Curcio, S. Salerno, G. Barbieri, L. De Bartolo, E. Drioli, and A. Bader, Biomaterials, 28, 5487 (2007).

    CAS  PubMed  Google Scholar 

  28. K. C. Murphy, B. P. Hung, S. Browne-Bourne, D. Zhou, J. Yeung, D. C. Genetos, and J. K. Leach, 14, 20160851 (2017).

  29. H. Ijima, T. Matsushita, K. Nakazawa, Y. Fujii, and K. Funatsu, Tissue Eng., 4, 213 (1998).

    CAS  Google Scholar 

  30. L. Xia, R. B. Sakban, Y. Qu, X. Hong, W. Zhang, B. Nugraha, W. H. Tong, A. Ananthanarayanan, B. Zheng, I. Y. Chau, R. Jia, M. McMillian, J. Silva, S. Dallas, and H. Yu, Biomaterials, 33, 2165 (2012).

    CAS  PubMed  Google Scholar 

  31. S. Yan, J. Wei, Y. Liu, H. Zhang, J. Chen, and X. Li, Acta Biomater., 28, 138 (2015).

    CAS  PubMed  Google Scholar 

  32. J. Sadoshima and S. Izumo, Annu. Rev. Physiol., 59, 551 (1997).

    CAS  PubMed  Google Scholar 

  33. Z. H. Syedain, J. S. Weinberg, and R. T. Tranquillo, Proc. Natl. Acad. Sci. U.S.A., 105, 6537 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. M. L. Funderburgh, M. M. Mann, and J. L. Funderburgh, Mol. Vis., 14, 308 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. U. Apte, V. Gkretsi, W. C. Bowen, W. M. Mars, J. H. Luo, S. Donthamsetty, A. Orr, S. P. Monga, C. Wu, & G. K. Michalopoulos, Hepatology (Baltimore, Md.), 50, 844 (2009).

    CAS  Google Scholar 

  36. J. S. Hammond, T. W. Gilbert, D. Howard, A. Zaitoun, G. Michalopoulos, K. M. Shakesheff, I. J. Beckingham, and S. F. Badylak, J. Hepatol., 54, 279 (2011).

    CAS  PubMed  Google Scholar 

  37. J. I. Jun and L. F. Lau, Aging, 2, 627 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. B. C. Tai, C. Du, S. Gao, A. C. Wan, and J. Y. Ying, Biomaterials, 31, 48 (2010).

    CAS  PubMed  Google Scholar 

  39. R. Lang, M. M, Stern, L. Smith, Y. Liu, S. Bharadwaj, G. Liu, P. M. Baptista, C. R. Bergman, S. Soker, J. J. Yoo, A. Atala, and Y. Zhang, Biomaterials, 32, 7042 (2011).

    CAS  PubMed  Google Scholar 

  40. P. Godoy, J. G. Hengstler, I. Ilkavets, C. Meyer, A. Bachmann, A. Muller, G. Tuschl, S. O. Mueller, and S. Dooley, Hepatology (Baltimore, Md.), 49, 2031 (2009).

    CAS  Google Scholar 

  41. M. H. Grant, M. D. Burke, G. M. Hawksworth, S. J. Duthie, J. Engeset, and J. C. Petrie, Biochem. Pharmacol., 36, 2311 (1987).

    CAS  PubMed  Google Scholar 

  42. M. Bernardi, C. S. Ricci, and G. Zaccherini, J. Clin. Exp. Hepatol., 4, 302 (2014).

    PubMed  PubMed Central  Google Scholar 

  43. M. Bernardi, C. Maggioli, and G. Zaccherini, Critical Care, 16, 211 (2012).

    PubMed  PubMed Central  Google Scholar 

  44. D. S. Dimski, J. Vet. Int. Med., 8, 73 (1994).

    CAS  Google Scholar 

  45. T. Takeda, S. Murphy, S. Uyama, G. M. Organ, B. L. Schloo, and J. P. Vacanti, Tissue Eng., 1, 253 (1995).

    CAS  PubMed  Google Scholar 

  46. M. Ito, H. Nagata, T. Yamamoto, D. Yoshihara, I. J. Fox, and S. Miyakawa, Cell Transpl., 16, 547 (2007).

    Google Scholar 

  47. J. Meyburg, F. Hoerster, J. Schmidt, J. Poeschl, G. F. Hoffmann, and J. P. Schenk, Cell Transpl., 19, 629 (2010).

    Google Scholar 

  48. I. J. Fox, J. R. Chowdhury, S. S. Kaufman, T. C. Goertzen, N. R. Chowdhury, P. I. Warkentin, K. Dorko, B. V. Sauter, and S. C. Strom, N. Engl. J. Med., 338, 1422 (1998).

    CAS  PubMed  Google Scholar 

  49. A. Dhawan, R. R. Mitry, R. D. Hughes, S. Lehec, C. Terry, S. Bansal, R. Arya, J. J. Wade, A. Verma, N. D. Heaton, M. Rela, and G. Mieli-Vergani, Transplantation, 78, 1812 (2004).

    PubMed  Google Scholar 

  50. M. Muraca, G. Gerunda, D. Neri, M. T. Vilei, A. Granato, P. Feltracco, M. Meroni, G. Giron, and A. B. Burlina, Lancet (London, England), 359, 317 (2002).

    Google Scholar 

  51. K. Tatsumi and T. Okano, Curr. Transplant. Rep., 4, 184 (2017).

    PubMed  PubMed Central  Google Scholar 

  52. E. K. Gustafson, G. Elgue, R. D. Hughes, R. R. Mitry, J. Sanchez, U. Haglund, S. Meurling, A. Dhawan, O. Korsgren, and B. Nilsson, Transplantation, 91, 632 (2011).

    CAS  PubMed  Google Scholar 

  53. C. A. Lee, A. Dhawan, R. A. Smith, R. R. Mitry, and E. Fitzpatrick, Cell Transplant., 25, 1227 (2016).

    PubMed  Google Scholar 

  54. A. Bader, E. Knop, A. Kern, K. Boker, N. Fruhauf, O. Crome, H. Esselmann, C. Pape, G. Kempka, and K. F. Sewing, Exp. Cell. Res., 226, 223 (1996).

    CAS  PubMed  Google Scholar 

  55. S. J. Peters, T. Vanhaecke, P. Papeleu, V. Rogiers, H. P. Haagsman, and K. Norren, Cell Tissue Res., 340, 451 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. J. C. Gerlach, K. Zeilinger, G. Spatkowski, F. Hentschel, N. Schnoy, S. Kolbeck, R. K. Schindler, and P. Neuhaus, J. Surg. Res., 100, 39 (2001).

    CAS  PubMed  Google Scholar 

  57. S. Jockenhoevel, G. Zund, S. P. Hoerstrup, K. Chalabi, J. S. Sachweh, L. Demircan, B. J. Messmer, and M. Turina, Eur. J. Cardio-thorac. Surg., 19, 424 (2001).

    CAS  Google Scholar 

  58. H. W. Kang, Y. Tabata, and Y. Ikada, Biomaterials, 20, 1339 (1999).

    CAS  PubMed  Google Scholar 

  59. I. R. Schwab, Trans. Am. Ophthalmol. Soc., 97, 891 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. C. Siltanen, M. Diakatou, J. Lowen, A. Haque, A. Rahimian, G. Stybayeva, and A. Revzin, Acta Biomater., 50, 428 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Author notes

  1. These authors contributed equally to the study.

Authors and Affiliations

  1. College of Pharmacy, Yeungnam University, Gyeongsan, Gyeongbuk, 38541, Korea

    Dong-Ho Ha, Phuong Mai Thi, Prerna Chaudhary & Jee-Heon Jeong

Authors
  1. Dong-Ho Ha
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Phuong Mai Thi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Prerna Chaudhary
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jee-Heon Jeong
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jee-Heon Jeong.

Additional information

Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Acknowledgments: This work was supported by the Yeungnam University Research Grant (2018).

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ha, DH., Thi, P.M., Chaudhary, P. et al. Efficient Formation of Three Dimensional Spheroids of Primary Hepatocytes Using Micropatterned Multi-Well Plates. Macromol. Res. 27, 938–943 (2019). https://doi.org/10.1007/s13233-019-7103-7

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13233-019-7103-7

Keywords

Search

Navigation