Advertisement

Encapsulation of Human Articular Chondrocytes into 3D Hydrogel: Phenotype and Genotype Characterization

  • Rui C. PereiraEmail author
  • Chiara Gentili
  • Ranieri Cancedda
  • Helena S. Azevedo
  • Rui L. Reis
Part of the Methods in Molecular Biology book series (MIMB, volume 695)

Abstract

This chapter is intended to provide a summary of the current materials used in cell encapsulation technology as well as methods for evaluating the performance of cells encapsulated in a polymeric matrix. In particular, it describes the experimental procedure to prepare a hydrogel matrix based on natural polymers for encapsulating and culturing human articular chondrocytes with the interest in cartilage regeneration. Protocols to evaluate the viability, proliferation, differentiation, and matrix production of embedded cells are also described and include standard protocols such as the MTT and [3H] Thymidine assays, reverse transcription polymerase chain reaction (RT-PCR) technique, histology, and immunohistochemistry analysis. The assessment of cell distribution within the 3D hydrogel construct is also described using APoTome analysis.

Key words

Natural polymers Hydrogels Gelation Chondrocyte encapsulation Cell culture and survival within 3D matrix Cell-based technology Cartilage regeneration 

Notes

Acknowledgements

The authors would like to thank Recco Orthopedic staff members for helpful discussions, particularly to Dr. Michele Grandizio; patients for biopsy material donation, as well as to Mrs Daniela Marubbi and Monica Scaranari for technical assistance with histological sample processing. This work was supported by funds from the Italian MUR (FIRB-Tissuenet project), the European Union funded STREP Project HIPPOCRATES (NMP3-CT-2003-505758), and the European NoE EXPERTISSUES (NMP3-CT-2004-500283).

References

  1. 1.
    Orive, G., Hernandez, R.M., Gascon, A.R., Calafiore, R., Chang, T.M.S., de Vos, P., Hortelano, G., Hunkeler, D., Lacik, I., and Pedraz, J.L. (2004) History, challenges and perspectives of cell microencapsulation. Trends Biotechnol. 22, 87–92.PubMedCrossRefGoogle Scholar
  2. 2.
    Lee, K.Y. and Mooney, D.J. (2001) Hydrogels for tissue engineering. Chem. Rev. 101, 1869–1879.PubMedCrossRefGoogle Scholar
  3. 3.
    Prokop, A., Hunkeler, D., DiMari, S., Haralson, M.A., and Wang, T.G. (1998) Water soluble polymers for immunoisolation I: Complex coacervation and cytotoxicity. Microencapsul. Microgels Iniferters 136, 1–51.CrossRefGoogle Scholar
  4. 4.
    Prokop, A., Hunkeler, D., Powers, A.C., Whitesell, R.R., and Wang, T.G. (1998) Water soluble polymers for immunoisolation II: Evaluation of multicomponent microencapsulation systems. Microencapsul. Microgels Iniferters 136, 53–73.CrossRefGoogle Scholar
  5. 5.
    Oliveira, J.T. and Reis, R.L., Hydrogels from polysaccharide-based materials: fundamentals and applications in regenerative medicine, in Natural-based polymers for biomedical applications, R.L. Reis, et al., Editors. 2008, Woodhead Publishing Limited: Cambridge. pp. 485–514.CrossRefGoogle Scholar
  6. 6.
    Yuguchi, Y., Thuy, T.T.T., Urakawa, H., and Kajiwara, K. (2002) Structural characteristics of carrageenan gels: temperature and concentration dependence. Food Hydrocoll. 16, 515–522.CrossRefGoogle Scholar
  7. 7.
    Rinaudo, M. (2008) Main properties and current applications of some polysaccharides as biomaterials. Polym. Int. 57, 397–430.CrossRefGoogle Scholar
  8. 8.
    Malpeli, M., Randazzo, N., Cancedda, R., and Dozin, B. (2004) Serum-free growth medium sustains commitment of human articular chondrocyte through maintenance of Sox9 expression. Tissue Eng. 10, 145–155.PubMedCrossRefGoogle Scholar
  9. 9.
    Johnstone, B., Hering, T.M., Caplan, A.I., Goldberg, V.M., and Yoo, J.U. (1998) In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp. Cell Res. 238, 265–272.PubMedCrossRefGoogle Scholar
  10. 10.
    Denizot, F. and Lang, R. (1986) Rapid colorimetric assay for cell-growth and survival – modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J. Immunol. Methods 89, 271–277.PubMedCrossRefGoogle Scholar
  11. 11.
    Yan, W.Q., Yang, T.S., Hou, L.Z., Suzuki, F., and Kato, Y. (1994) Effect of concanavalin A on morphology and DNA synthesis of resting chondrocyte cultures. Shi Yan Sheng Wu Xue Bao 27, 300–305.PubMedGoogle Scholar
  12. 12.
    Osakada, F., Ikeda, H., Mandai, M., Wataya, T., Watanabe, K., Yoshimura, N., Akaike, A., Sasai, Y., and Takahashi, M. (2008) The generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells. Nat. Biotechnol. 26, 352–352.CrossRefGoogle Scholar
  13. 13.
    Frey, M.R., Dise, R.S., Edelblum, K.L., and Polk, D.B. (2006) p38 kinase regulates epidermal growth factor receptor downregulation and cellular migration. EMBO J. 25, 5683–5692.PubMedCrossRefGoogle Scholar
  14. 14.
    Pereira, R.C., Scaranari, M., Castagnola, P., Grandizio, M., Azevedo, H.S., Reis, R.L., Cancedda, R., and Gentili, C. (2009) Novel injectable gel (system) as a vehicle for human articular chondrocytes in cartilage tissue regeneration. J. Tissue Eng. Regen. Med. 3, 97–106.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Rui C. Pereira
    • 1
    • 2
    • 3
    Email author
  • Chiara Gentili
    • 3
  • Ranieri Cancedda
    • 3
  • Helena S. Azevedo
    • 1
    • 2
  • Rui L. Reis
    • 1
    • 2
  1. 1.Biomaterials, Biodegradables and BiomimeticsUniversity of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative MedicineGuimarãesPortugal
  2. 2.PTGovernment Associated LaboratoryInstitute for Biotechnology and BioengineeringGuimarãesPortugal
  3. 3.Dipartimento di Biologia, Oncologia e GeneticaUniversita di Genoval & Istituto Nazionale per la Ricerca sul CancroGenovaItaly

Personalised recommendations