Colloid and Polymer Science

, Volume 292, Issue 4, pp 839–848 | Cite as

Highly robust protein production by co-culture of CHO spheroids layered on feeder cells in serum-free medium

  • Koichi Kutsuzawa
  • Toshihiro Suzuki
  • Hidehiro Kishimoto
  • Akiichi Murakami
  • Takachika Azuma
  • Ryo Abe
  • Hidenori Otsuka
Original Contribution
First Online:
Received:
Revised:
Accepted:

Abstract

Recombinant Chinese hamster ovary (rCHO) cells have been the most commonly used mammalian host for large-scale commercial production of therapeutic proteins. Although recent advances in 3D culture of rCHO cells is preferred to 2D monolayer culture for highly productive and robust expression of therapeutic proteins, there exists still limitation for efficient protein production. Therefore, a new cell culture system is essentially required for an efficient protein production. Here, we report on a new 3D cell culture system as a spheroid cell culture on the micropattern array for efficient production of protein by CHO cells. Particularly, cocultivation of CHO spheroids with bovine aortic endothelial cells (BAEC) as a feeder layer cells was essential to stably increase a protein production. We investigated the co-culture mechanism of functional enhancement with respect to the cell–cell interactions. Functional comparison between 2D and 3D co-cultures suggested the preferred configuration as spheroid for higher protein production. Specifically, to estimate the effect of respective cell constitution in co-cultured spheroids on the protein production per CHO cell, the number of viable cells in cell proliferation was determined with culture periods. These studies demonstrated the significant role of micropatterned BAEC as a feeder layer for the retained formation of CHO spheroids, resulting in predominantly enhanced production of proteins, although the functional enhancement of CHO cells was obtained by co-culture with BAECs in both 2D and 3D configurations. Thus, heterotypic cell communications that play indispensable roles in increasing CHO functions should be properly obtained in 3D cell configurations. Significantly, these spheroids in the serum-free medium drastically enhanced protein expression level up to sevenfold compared with CHO monospheroids, suggesting that a suitable culture conditions for heterotypic cell–cell interactions would allow improved protein secretion to occur unimpeded.

Keywords

Recombinant therapeutic proteins Chinese hamster ovary (CHO) cells Spheroid 3D co-culture 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgments

The present study was supported in part by KAKENHI(22700479), “Grant-in-Aid for Young Scientists(B)”, JST, CREST, and Grant-in-Aid for Fundamental Research Project for Autonomous Regeneration Device from New Energy and Industrial Technology Development Organization, Japan. This work was also supported in part by Ministry of Education, Culture, Sports, Science and Technology Grants S0901020.

References

  1. 1.
    Wurm FM (2004) Nat Biotechnol 11:1393–1398CrossRefGoogle Scholar
  2. 2.
    Gerngross TU (2004) Nat Biotechnol 11:409–414Google Scholar
  3. 3.
    Chusainow J, Yang YS, Yeo JHM, Toh PC, Asvadi P, Wong NSC, Yap MGS (2008) Biotechnol Bioeng 102(4):1182–1196CrossRefGoogle Scholar
  4. 4.
    Pilbrough W, Munro TP, Gray P (2009) PLoS One 4(12):e8432–e8441CrossRefGoogle Scholar
  5. 5.
    Chennamsettya N, Voynova V, Kaysera V, Helkb B, Trouta BL (2009) Proc Natl Acad Sci U S A 106:11937–11942CrossRefGoogle Scholar
  6. 6.
    Perchiacca JM, Ladiwala ARA, Bhattacharya M, Tessier PM (2012) Proc Natl Acad Sci USA 109:84–89CrossRefGoogle Scholar
  7. 7.
    Mohan C, Kim YG, Koo J, Lee GM (2008) Biotechnol J 5:624–630CrossRefGoogle Scholar
  8. 8.
    Gottschalk U, Brorson K, Shukla AA (2012) Nat Biotechnol 30:489–492CrossRefGoogle Scholar
  9. 9.
    Kim JY, Kim YG, Lee GM (2012) Appl Microbiol Biotechnol 93:917–930CrossRefGoogle Scholar
  10. 10.
    Girod PA, Lefourn V, Calabrese D, Regamey A, Claude A, Gaussian A, Fisch I (2012) BioProcess International 10(1):58–61Google Scholar
  11. 11.
    Otsuka H, Hirano A, Nagasaki Y, Okano T, Horiike Y, Kataoka K (2004) ChemBioChem 5:850–855CrossRefGoogle Scholar
  12. 12.
    Otsuka H (2010) Molecules 15:5525–5546CrossRefGoogle Scholar
  13. 13.
    Nakasone Y, Yamamoto M, Tateishi T, Otsuka H (2011) IEICE TRANS ELECTRON E94-C(2):176–180CrossRefGoogle Scholar
  14. 14.
    Kutsuzawa K, Takahashi C, Sato S, Suzuki T, Kishimoto H, Murakami A, Azuma T, Abe R, Otsuka H (2013) J Nanosci Nanotechnol 13:229–235CrossRefGoogle Scholar
  15. 15.
    Michalopoulos GK, DeFrances MC (1997) Science 276:60–66CrossRefGoogle Scholar
  16. 16.
    Cho CH, Berthiaume F, Tilles AW, Yarmush ML (2008) Biotechnol Bioeng 101:345–356CrossRefGoogle Scholar
  17. 17.
    van Poll D, Parekkadan B, Cho CH, Berthiaume F, Nahmias Y, Tilles AW, Yarmush ML (2008) Hepatology 47:1634–1643CrossRefGoogle Scholar
  18. 18.
    Dunn JC, Tompkins RG, Yarmush ML (1992) J Cell Biol 116:1043–1053CrossRefGoogle Scholar
  19. 19.
    Sanchez-Bustamante CD, Kelm JM, Mitta B, Fussenegger M (2006) Biotechnol Bioeng 93:169–180CrossRefGoogle Scholar
  20. 20.
    Stahl A, Wenger A, Weber H, Stark GB, Augustin HG, Finkenzeller G (2004) Biochem Biophys Res Commun 322:684–692CrossRefGoogle Scholar
  21. 21.
    Azuma K, Nagaoka M, Cho CS, Akaike T (2010) Biomaterials 5:802–809CrossRefGoogle Scholar
  22. 22.
    Capon DJ, Chamow SM, Mordenti J, Marsters SA, Gregory T, Mitsuya H, Byrn RA, Lucas C, Wurm FM, Groopman JE, Broder S, Smith DH (1989) Nature 337:525–531CrossRefGoogle Scholar
  23. 23.
    Traunecker A, Schneider J, Kiefer H, Karjalainen K (1989) Nature 339:68–70CrossRefGoogle Scholar
  24. 24.
    Satomi T, Nagasaki Y, Kobayashi H, Otsuka H, Kataoka K (2007) Langmuir 23:6698–6703CrossRefGoogle Scholar
  25. 25.
    Uchida K, Hoshino Y, Tamura A, Yoshimoto K, Kojima S, Yamashita K, Yamanaka I, Otsuka H, Kataoka K, Nagasaki Y (2007) Biointerphases 2:126–130CrossRefGoogle Scholar
  26. 26.
    Rhim JA, Sandgren EP, Degan JL, Palmiter RD, Brinster RL (1994) Science 263:1149–1152CrossRefGoogle Scholar
  27. 27.
    Chia SM, Lin PC, Yu H (2005) Biotechnol Bioeng 89(5):565–573CrossRefGoogle Scholar
  28. 28.
    Ohashi K, Yokoyama T, Yamato M, Kuge H, Kanehiro H, Tsutsumi M, Amanuma T, Iwata H, Yang J, Okano T, Nakajima Y (2007) Nature Med 13:880–885CrossRefGoogle Scholar
  29. 29.
    Nahmias Y, Casali M, Barbe L, Berthiaume F, Yarmush ML (2006) Hepatology 43:257–265CrossRefGoogle Scholar
  30. 30.
    Matsumoto K, Yoshitomi H, Rossant J, Zaret KS (2001) Science 294:559–563CrossRefGoogle Scholar
  31. 31.
    Zaret KS (2002) Nature Rev Genet 3:499–512CrossRefGoogle Scholar
  32. 32.
    LeCouter J, Moritz DR, Li B, Phillips GL, Liang H, Gerber HP, Hillan KJ, Ferrara N (2003) Science 299:890–893CrossRefGoogle Scholar
  33. 33.
    Hui EE, Bhatia SN (2007) Proc Natl Acad Sci U S A 104:5722–5726CrossRefGoogle Scholar
  34. 34.
    Ito M, Taguchi T (2009) Acta Biomater 8:2945–2952CrossRefGoogle Scholar
  35. 35.
    Kunz-Schughart LA, Schroeder JA, Wondrak M, van Rey F, Lehle K, Hofstaedter F, Wheatley DN (2006) Am J Physiol Cell Physiol 290:C1385–C1398CrossRefGoogle Scholar
  36. 36.
    Wang C, Roy SK (2010) Endocrinology 151(5):2319–2330CrossRefGoogle Scholar
  37. 37.
    Khetani SR, Szulgit G, Rio JAD, Barlow C, Bhatia SN (2004) Hepatology 40:545–554CrossRefGoogle Scholar
  38. 38.
    Brieva T, Moghe P (2004) Tissue Eng 10:553–564CrossRefGoogle Scholar
  39. 39.
    Yamane A, Seetharam L, Yamaguchi S, Gotoh N, Takahashi T, Neufeld G, Shibuya M (1994) Oncogene 9:2683–2690Google Scholar
  40. 40.
    Tsuda Y, Kikuchi A, Yamato M, Chen G, Okano T (2006) Biochem Biophys Res Commun 348:937–944CrossRefGoogle Scholar
  41. 41.
    Schröder M, Matischak K, Friedl P (2004) J Biotechnol 108:279–292CrossRefGoogle Scholar
  42. 42.
    Skelton D, Goodyear A, Ni D, Walton WJ, Rolle M, Hare JT, Logan TM (2010) J Biomol NMR:93–102.Google Scholar
  43. 43.
    Arthuso FS, Bartolini P, Soares CRJ (2012) Appl Biochem Biotechnol 167:2212–2224CrossRefGoogle Scholar
  44. 44.
    Kamoda S, Nomura C, Kinoshita M, Nishiura S, Ishikawa R, Kakehi K, Kawasaki N, Hayakawa T (2004) J Chromatogr A 1050:211–216CrossRefGoogle Scholar
  45. 45.
    Kanda Y, Yamane-Ohnuki N, Sakai N, Yamano K, Nakano R, Inoue M, Misaka H, Iida S, Wakitani M, Konno Y, Yano K, Shitara K, Hosoi S, Satoh M (2006) Biotechnol Bioeng 94:680–688CrossRefGoogle Scholar
  46. 46.
    Dietmair S, Hodson MP, Quek LE, Timmins NE, Chrysanthopoulos P, Jacob SS, Gray P, Nielsen LK (2012) Biotechnol Bioeng 109:1404–1414CrossRefGoogle Scholar
  47. 47.
    Costa AR, Rodrigues EM, Henriques M, Azeredo J, Oliveira R (2010) Eur J Pharm Biopharm 74:127–138CrossRefGoogle Scholar
  48. 48.
    Glassy MC, Oeters RE, Mikhaley A (1987) In Vitro Cell Dev Biol 23(11):745–749CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Koichi Kutsuzawa
    • 1
    • 2
  • Toshihiro Suzuki
    • 3
    • 5
  • Hidehiro Kishimoto
    • 3
    • 5
  • Akiichi Murakami
    • 4
  • Takachika Azuma
    • 4
    • 5
  • Ryo Abe
    • 3
    • 5
  • Hidenori Otsuka
    • 1
    • 2
    • 5
  1. 1.Department of Applied Chemistry, Faculy of ScienceTokyo University of ScienceShinjuku-kuJapan
  2. 2.Center for Colloid and Interface Science, Research Institute for Science and TechnologyTokyo University of ScienceShinjuku-kuJapan
  3. 3.Division of Immunobiology, Research Institute for Biomedical SciencesTokyo University of ScienceChibaJapan
  4. 4.Division of Biosignaling, Research Institute for Biomedical SciencesTokyo University of ScienceChibaJapan
  5. 5.Center for Technologies against Cancer, Research Institute for Science and TechnologyTokyo University of ScienceChibaJapan

Personalised recommendations