Cytotechnology

, Volume 63, Issue 6, pp 633–643 | Cite as

Derivation, characterization and expansion of fetal chondrocytes on different microcarriers

  • Gaye Çetinkaya
  • Anıl Sera Kahraman
  • Menemşe Gümüşderelioğlu
  • Sezen Arat
  • Mehmet Ali Onur
Original Research

Abstract

Fetal chondrocytes (FCs) have recently been identified as an alternative cell source for cartilage tissue engineering applications because of their partially chondrogenically differentiated phenotype and developmental plasticity. In this study, chondrocytes derived from fetal bovine cartilage were characterized and then cultured on commercially available Cytodex-1 and Biosilon microcarriers and thermosensitive poly(hydroxyethylmethacrylate)-poly(N-isopropylacrylamide) (PHEMA-PNIPAAm) beads produced by us. Growth kinetics of FCs were estimated by means of specific growth rate and metabolic activity assay. Cell detachment from thermosensitive microcarriers was induced by cold treatment at 4 °C for 20 min or enzymatic treatment was applied for the detachment of cells from Cytodex-1 and Biosilon. Although attachment efficiency and proliferation of FCs on PHEMA-PNIPAAm beads were lower than that of commercial Cytodex-1 and Biosilon microcarriers, these beads also supported growth of FCs. Detached cells from thermosensitive beads by cold induction exhibited a normal proliferative activity. Our results indicated that Cytodex-1 microcarrier was the most suitable material for the production of FCs in high capacity, however, ‘thermosensitive microcarrier model’ could be considered as an attractive solution to the process scale up for cartilage tissue engineering by improving surface characteristics of PHEMA-PNIPAAm beads.

Keywords

Fetal chondrocyte Thermosensitive polymers Microcarriers PNIPAAm Cartilage tissue engineering 

References

  1. Anghileri LJ, Dermietzel R (1976) Cell coat in tumour cells—effects of trypsin and EDTA: a biochemical and morphological study. Oncology 33:17–23CrossRefGoogle Scholar
  2. Au A, Ha J, Polotsky A, Krzyminski K, Gutowska A, Hungerford DS, Frondoza CG (2003) Thermally reversible polymer gel for chondrocyte culture. J Biomed Mater Res A 67:1310–1319CrossRefGoogle Scholar
  3. Baker T, Goodwin T (1997) Three-dimensional culture of bovine chondrocytes in rotating-wall vessels. In Vitro Cell Dev Biol Anim 33:358–365CrossRefGoogle Scholar
  4. Benya PD, Shaffer JD (1982) Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell 30:215–224CrossRefGoogle Scholar
  5. Bouchet B, Colon M, Polotsky A, Shikani AH, Hungerford DS, Frondoza CG (2000) Beta-1 integrin expression by human nasal chondrocytes in microcarrier spinner culture. J Biomed Mater Res 52:716–724CrossRefGoogle Scholar
  6. Canavan HE, Cheng X, Graham DJ, Ratner BD, Castner DG (2005) Cell sheet detachment affects the extracellular matrix: a surface science study comparing thermal liftoff, enzymatic, and mechanical methods. J Biomed Mater Res A 75:1–13Google Scholar
  7. Chen JP, Cheng TH (2006) Thermoresponsive chitosan-graft-poly(N-isopropylacrylamide) injectable hydrogel for cultivation of chondrocytes and meniscus cells. Macromol Biosci 6:1026–1039CrossRefGoogle Scholar
  8. Dowthwaite GP, Bishop JC, Redman SN, Khan IM, Rooney P, Evans DJR, Haughton L, Bayram Z, Boyer S, Thomson B, Wolfe MS, Archer CW (2004) The surface of articular cartilage contains a progenitor cell population. J Cell Sci 117:889–897CrossRefGoogle Scholar
  9. Freed LE, Vunjak-Novakovic G, Langer R (1993) Cultivation of cell–polymer cartilage implants in bioreactors. J Cell Biochem 51:257–264CrossRefGoogle Scholar
  10. Freshney IR (2005) culture of animal cells a manual of basic technique, 5th edn. Wiley, New York, p 208Google Scholar
  11. Frondoza C, Sohrabi A, Hungerford D (1996) Human chondrocytes proliferate and produce matrix components in microcarrier suspension culture. Biomaterials 17:879–888CrossRefGoogle Scholar
  12. Fujioka N, Morimoto Y, Takeuchi K, Yoshioka M, Kikuchi M (2003) Difference in infrared spectra from cultured cells dependent on cell-harvesting method. Appl Spectro 57:241–243CrossRefGoogle Scholar
  13. Giard D (1986) Detachment of anchorage dependent cells from microcarriers, World Intellectual Property Organization International Bureau WO 86/01531Google Scholar
  14. Gümüşderelioğlu M (2011) Development of temperature-sensistive microcarriers for large scale cell cultures, Turkish Scientific and Research Council Project (109M228) ReportGoogle Scholar
  15. GE Healthcare (2005) Microcarrier cell culture: principles and methods. General Electric CompanyGoogle Scholar
  16. Jasionowski M, Kryminski K, Chrisler W, Markille LM, Morris J, Gutowska A (2004) Thermally-reversible gel for 3-D cell culture of chondrocytes. J Mat Sci Mater Med 15:575–582CrossRefGoogle Scholar
  17. Jung K, Hampel G, Scholz M, Henke W (1995) Culture of human kidney proximal tubular cells—the effect of various detachment procedures on viability and degree of cell detachment. Cell Physiol Biochem 5:353–360CrossRefGoogle Scholar
  18. Kim MR, Jeong JH, Park TK (2002) Swelling induced detachment of chondrocytes using RGD-modified poly(N-isopropylacrylamide) hydrogel beads. Biotechnol Prog 18:495–500CrossRefGoogle Scholar
  19. Kim DJ, Heo J-y, Kim KS, Choi IS (2003) Formation of thermoresponsive poly(N-isopropylacrylamide)/dextran particles by atom transfer radical polymerization. Macromol Rapid Commun 24(8):517–521CrossRefGoogle Scholar
  20. Kiremitçi M, Çukurova H (1992) Production of highly crosslinked hydrophilic polymer beads:effect of polymerization conditions on particle size and size distribution. Polymer 33(15):3257–3261CrossRefGoogle Scholar
  21. Lopes AAB, Peranovich TMS, Maeda NY, Bydlowski SP (2001) Differential effects of enzymatic treatments on the storage and secretion of von Willebrand factor by human endothelial cells. Thromb Res 101:291–297CrossRefGoogle Scholar
  22. Mahmoudifar N, Doran PM (2005) Tissue engineering of human cartilage and osteochondral composites using recirculation bioreactors. Biomaterials 34:7012–7024CrossRefGoogle Scholar
  23. Mahmoudifar N, Doran PM (2006) Effect of seeding and bioreactor culture conditions on the development of human tissue-engineered cartilage. Tissue Eng 12:1675–1685CrossRefGoogle Scholar
  24. Malda J, Frondoza CG (2006) Microcarriers in the engineering of cartilage and bone. Trends Biotechnol 7:299–304CrossRefGoogle Scholar
  25. Malda J, Kreijveld E, Temenoff JS, van Blitterswijk CA, Riesle J (2003) Expansion of bovine chondrocytes on microcarriers enhances redifferentiation. Tissue Eng 9:939–948CrossRefGoogle Scholar
  26. Martin JM, Smith M, Al-Rubeai M (2005) Cryopreservation and in vitro expansion of chondroprogenitor cells isolated from the superficial zone of articular cartilage. Biotechnol Prog 21:168–177CrossRefGoogle Scholar
  27. Melero-Martin JM, Dowling MA, Smith M, Al-Rubeai M (2006) Expansion of chondroprogenitor cells on macroporous microcarriers as an alternative to conventional monolayer systems. Biomaterials 27:2970–2979CrossRefGoogle Scholar
  28. Montjovent MO, Bocelli-Tyndall C, Scaletta C, Scherberich A, Mark S, Martin I, Applegate LA, Pioletti DP (2009) In vitro characterization of immune-related properties of human fetal bone cells for potential tissue engineering applications. Tissue Eng Part A 15:1523–1532CrossRefGoogle Scholar
  29. Parsch D, Brummendorf TH, Richter W, Fellenberg J (2002) Replicative aging of human articular chondrocytes during ex vivo expansion. Arthritis Rheum 46:2911–2916CrossRefGoogle Scholar
  30. Pioletti DP, Montjovent MO, Zambelli PY, Applegate L (2006) Bone tissue engineering using foetal cell therapy. Swiss Med Wkly 136:557–560Google Scholar
  31. Quintin A, Schizas C, Scaletta C, Jaccoud S, Applegate LA, Pioletti DP (2010) Plasticity of fetal cartilaginous cells. Cell Transpl 19:1349–1357CrossRefGoogle Scholar
  32. Reiners JJ, Mathieu P, Okafor C, Putt DA, Lash L (2000) Depletion of cellular glutathione by conditions used for the passaging of adherent cultured cells. Toxicol Lett 115:153–163CrossRefGoogle Scholar
  33. Silva R, Mano JF, Reis RL (2007) Smart thermoresponsive coatings and surfaces for tissue engineering: switching cell-material boundaries. Trends Biotechnol 25:577–583CrossRefGoogle Scholar
  34. Umegaki R, Masahiro KO, Taya M (2004) Assessment of cell detachment and growth potential of human keratinocyte based on observed changes in individual cell area during trypsinization. Biochem Eng J 17:49–55CrossRefGoogle Scholar
  35. Varani J, Dame M, Rediske J, Beals TF, Hillegas W (1985) Substrate-dependent differences in growth and biological properties of fibroblasts and epithelial cells grown in microcarrier culture. J Biol Stand 13:67–76CrossRefGoogle Scholar
  36. Varani J, Bnedelow M, Chun JH, Hillegas WA (1986) Cell growh on microcarriers: comparison of proliferation on and recovery from various substrates. J Biol Stand 14:331–336CrossRefGoogle Scholar
  37. Von der Mark K, Gauss V, von der Mark H, Mueller P (1977) Relationship between cell shape and type of collagen synthesized as chondrocytes lose their cartilage phenotype in culture. Nature 267:531–532CrossRefGoogle Scholar
  38. Weber C, Pohl S, Portner R, Wallrapp C, Kassem M, Geigle P, Czermak P (2007) Expansion and harvesting of hMSC-TERT. Open Biomed Eng J 1:38–46Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Gaye Çetinkaya
    • 1
  • Anıl Sera Kahraman
    • 2
  • Menemşe Gümüşderelioğlu
    • 2
  • Sezen Arat
    • 1
  • Mehmet Ali Onur
    • 3
  1. 1.TUBITAK MRC-Genetic Engineering Biotechnology Institute (GEBI)Gebze/KocaeliTurkey
  2. 2.Chemical Engineering and Bioengineering DepartmentsHacettepe UniversityBeytepeTurkey
  3. 3.Faculty of Science, Department of BiologyHacettepe UniversityBeytepeTurkey

Personalised recommendations