Skip to main content
SpringerLink
Log in
SpringerLink
Log in

3D Cell Culture: A Review of Current Approaches and Techniques

  • Protocol
  • First Online:
3D Cell Culture

Part of the book series: Methods in Molecular Biology ((MIMB,volume 695))

Abstract

Cell culture in two dimensions has been routinely and diligently undertaken in thousands of laboratories worldwide for the past four decades. However, the culture of cells in two dimensions is arguably primitive and does not reproduce the anatomy or physiology of a tissue for informative or useful study. Creating a third dimension for cell culture is clearly more relevant, but requires a multidisciplinary approach and multidisciplinary expertise. When entering the third dimension, investigators need to consider the design of scaffolds for supporting the organisation of cells or the use of bioreactors for controlling nutrient and waste product exchange. As 3D culture systems become more mature and relevant to human and animal physiology, the ability to design and develop co-cultures becomes possible as does the ability to integrate stem cells. The primary objectives for developing 3D cell culture systems vary widely – and range from engineering tissues for clinical delivery through to the development of models for drug screening. The intention of this review is to provide a general overview of the common approaches and techniques for designing 3D culture models.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Freshney, I. R. (2005) Culture of animal Cells. A Manual of Basic Technique, 5 ed. John Wiley & Sons, Hoboken, NJ.

    Book  Google Scholar 

  2. Abbott, A. (2003) Cell culture: Biology’s new dimension. Nature 424, 870–872.

    Article  PubMed  CAS  Google Scholar 

  3. Langer, R. and Tirrell, D. A. (2004) Designing materials for biology and medicine. Nature 428, 487–492.

    Article  PubMed  CAS  Google Scholar 

  4. Lee, J., Cuddihy, M. J., and Kotov, N. A. (2008) Three-dimensional cell culture matrices: State of the art. Tissue Eng. Part B Rev. 14, 61–86.

    Article  PubMed  CAS  Google Scholar 

  5. Lavik, E. and Langer, R. (2004) Tissue engineering: current state and perspectives. Applied Microbiol. Biotechnol. 65, 1–8.

    Article  CAS  Google Scholar 

  6. Pampaloni, F., Reynaud, E. G., and Stelzer, E. H. K. (2007) The third dimension bridges the gap between cell culture and live tissue. Nature Rev. Mol. Cell Biol. 8, 839–845.

    Article  CAS  Google Scholar 

  7. Toda, S., Watanabe, K., Yokoi, F., Matsumura, S., Suzuki, K., Ootani, A., Aoki, A., Koike, N., and Sugihara, H. (2002) A new organotypic culture of thyroid tissue maintains three-dimensional follicles with C cells for a long term. Biochem. Biophys. Res. Commun. 294, 906–911.

    Article  PubMed  CAS  Google Scholar 

  8. Hadjantonakis, A. K., Dickinson, M. E., Fraser, S. E., and Papaioannou, V. E. (2003) Technicolour transgenics: Imaging tools for functional genomics in the mouse. Nature Rev. Genet. 4, 613–625.

    Article  PubMed  CAS  Google Scholar 

  9. Timmins, N. E., Harding, F. J., Smart, C., Brown, M. A., and Nielsen, L. K. (2005) Method for the generation and cultivation of functional three-dimensional mammary constructs without exogenous extracellular matrix. Cell Tissue Res. 320, 207–210.

    Article  PubMed  CAS  Google Scholar 

  10. Castaneda, F. and Kinne, R. K. H. (2000) Short exposure to millimolar concentrations of ethanol induces apoptotic cell death in multicellular HepG2 spheroids. J. Cancer Res. Clin. Oncol. 126, 305–310.

    Article  PubMed  CAS  Google Scholar 

  11. Ivascu, A. and Kubbies, M. (2006) Rapid generation of single-tumor spheroids for high-throughput cell function and toxicity analysis. J. Biomol. Screen. 11, 922–932.

    Article  PubMed  CAS  Google Scholar 

  12. MacNeil, S. (2007) Progress and opportunities for tissue-engineered skin. Nature 445, 874–880.

    Article  PubMed  CAS  Google Scholar 

  13. Ghosh, M. M., Boyce, S., Layton, C., Freedlander, E., and MacNeil, S. (1997) A comparison of methodologies for the preparation of human epidermal-dermal composites. Annals Plastic Surg. 39, 390–404.

    Article  CAS  Google Scholar 

  14. Griffith, L. G. and Swartz, M. A. (2006) Capturing complex 3D tissue physiology in vitro. Nature Rev. Mol. Cell Biol. 7, 211–224.

    Article  CAS  Google Scholar 

  15. Canton, I., Sarwar, U., Kemp, E. H., Ryan, A. J., MacNeil, S., and Haycock, J. W. (2007) Real-time detection of stress in 3D tissue-engineered constructs using NF-kB activation in transiently transfected human dermal fibroblasts. Tissue Eng. 13, 1013–1024.

    Article  PubMed  CAS  Google Scholar 

  16. Allen, L. T., Tosetto, M., Miller, I. S., O’Connor, D. P., Penney, S. C., Lynch, I., Keenan, A. K., Pennington, S. R., Dawson, K. A., and Gallagher, W. M. (2006) Surface-induced changes in protein adsorption and implications for cellular phenotypic responses to surface interaction. Biomaterials 27, 3096–3108.

    Article  PubMed  CAS  Google Scholar 

  17. Wilson, C. J., Clegg, R. E., Leavesley, D. I., and Pearcy, M. J. (2005) Mediation of biomaterial-cell interactions by adsorbed proteins: A review. Tissue Eng. 11, 1–18.

    Article  PubMed  CAS  Google Scholar 

  18. Vroman, L. and Lukosevicius, A. (1964) Ellipsometer recordings of changes in optical thickness of adsorbed films associated with surface activation of blood clotting. Nature 204, 701.

    Article  PubMed  CAS  Google Scholar 

  19. Vroman, L. (1962) Effect of adsorbed proteins on wettability of hydrophilic and hydrophobic solids. Nature 196, 476.

    Article  PubMed  CAS  Google Scholar 

  20. Murray-Dunning, C. M., McKean, R., Forster, S., Ryan, A. J., McArthur, S. L., and Haycock, J. W. (2010) Three-dimensional alignment of Schwann cells using hydrolysable microfibre scaffolds – strategies for peripheral nerve repair. Methods Mol. Biol. 695, 155–166.

    Google Scholar 

  21. France, R. M., Short, R. D., Dawson, R. A., and MacNeil, S. (1998) Attachment of human keratinocytes to plasma co-polymers of acrylic acid octa-1,7-diene and allyl amine octa-1,7-diene. J. Mat. Chem. 8, 37–42.

    Article  CAS  Google Scholar 

  22. Sharma, S., Johnson, R. W., and Desai, T. A. (2004) Evaluation of the stability of nonfouling ultrathin poly(ethylene glycol) films for silicon-based microdevices. Langmuir 20, 348–356.

    Article  PubMed  CAS  Google Scholar 

  23. Massia, S. P. and Hubbell, J. A. (1991) An RGD spacing of 440 nM is sufficient for integrin alpha-v-beta-3-mediated fibroblast spreading and 140 nM for focal contact and stress fiber formation. J. Cell Biol. 114, 1089–1100.

    Article  PubMed  CAS  Google Scholar 

  24. Barry, J. J. A., Silva, M. M. C. G., Shakesheff, K. M., Howdle, S. M., and Alexander, M. R. (2005) Using plasma deposits to promote cell population of the porous interior of three-dimensional poly(d,l-lactic acid) tissue-engineering scaffolds. Adv. Funct. Mater. 15, 1134–1140.

    Article  CAS  Google Scholar 

  25. Hollister, S. J. (2005) Porous scaffold design for tissue engineering. Nature Mater. 4, 518–524.

    Article  CAS  Google Scholar 

  26. Sun, T., Norton, D., Vickers, N., McArthur, S. L., Mac Neil, S., Ryan, A. J., and Haycock, J. W. (2008) Development of a bioreactor for evaluating novel nerve conduits. Biotechnol. Bioeng. 99, 1250–1260.

    Article  PubMed  CAS  Google Scholar 

  27. Sun, T., Mai, S. M., Norton, D., Haycock, J. W., Ryan, A. J., and MacNeil, S. (2005) Self-organization of skin cells in three-dimensional electrospun polystyrene scaffolds. Tissue Eng. 11, 1023–1033.

    Article  PubMed  CAS  Google Scholar 

  28. Blackwood, K. A., McKean, R., Canton, I., Freeman, C. O., Franklin, K. L., Cole, D., Brook, I., Farthing, P., Rimmer, S., Haycock, J. W., Ryan, A. J., and MacNeil, S. (2008) Development of biodegradable electrospun scaffolds for dermal replacement. Biomaterials 29, 3091–3104.

    Article  PubMed  CAS  Google Scholar 

  29. Stevens, M. M. and George, J. H. (2005) Exploring and engineering the cell surface interface. Science 310, 1135–1138.

    Article  PubMed  CAS  Google Scholar 

  30. Martin, I., Wendt, D., and Heberer, M. (2004) The role of bioreactors in tissue engineering. Trends Biotechnol. 22, 80–86.

    Article  PubMed  CAS  Google Scholar 

  31. Allori, A. C., Sailon, A. M., Pan, J. H., and Warren, S. M. (2008) Biological basis of bone formation, remodeling, and repair – Part III: Biomechanical forces. Tissue Eng. Part B Rev. 14, 285–293.

    Article  PubMed  CAS  Google Scholar 

  32. Engler, A. J., Sen, S., Sweeney, H. L., and Discher, D. E. (2006) Matrix elasticity directs stem cell lineage specification. Cell 126, 677–689.

    Article  PubMed  CAS  Google Scholar 

  33. Sun, T., Norton, D., Mckean, R. J., Haycock, J. W., Ryan, A. J., and MacNeil, S. (2007) Development of a 3D cell culture system for investigating cell interactions with electrospun fibers. Biotechnol. Bioeng. 97, 1318–1328.

    Article  PubMed  CAS  Google Scholar 

  34. Geiger, B., Spatz, J. P., and Bershadsky, A. D. (2009) Environmental sensing through focal adhesions. Nature Rev. Mol. Cell Biol. 10, 21–33.

    Article  CAS  Google Scholar 

  35. Zhang, S. (2008) Designer self-assembling peptide nanofiber scaffolds for study of 3-D cell biology and beyond. Adv. Cancer Res. 99, 335–340.

    Article  PubMed  CAS  Google Scholar 

  36. Ma, P. X. and Zhang, R. Y. (1999) Synthetic nano-scale fibrous extracellular matrix. J. Biomed. Mater. Res. 46, 60–72.

    Article  PubMed  CAS  Google Scholar 

  37. Sachlos, E., Gotora, D., and Czernuszka, J. T. (2006) Collagen scaffolds reinforced with biomimetic composite nano-sized carbonate-substituted hydroxyapatite crystals and shaped by rapid prototyping to contain internal microchannels. Tissue Eng. 12, 2479–2487.

    Article  PubMed  CAS  Google Scholar 

  38. VunjakNovakovic, G., Freed, L. E., Biron, R. J., and Langer, R. (1996) Effects of mixing on the composition and morphology of tissue-engineered cartilage. AIChE J. 42, 850–860.

    Article  CAS  Google Scholar 

  39. Unsworth, B. R. and Lelkes, P. I. (1998) Growing tissues in microgravity. Nature Med. 4, 901–907.

    Article  PubMed  CAS  Google Scholar 

  40. Wendt, D., Marsano, A., Jakob, M., Heberer, M., and Martin, I. (2003) Oscillating perfusion of cell suspensions through three-dimensional scaffolds enhances cell seeding efficiency and uniformity. Biotechnol. Bioeng. 84, 205–214.

    Article  PubMed  CAS  Google Scholar 

  41. Jasmund, I., Simmoteit, R., and Bader, A. (2001) An improved oxygenation hollow fiber bioreactor for the cultivation of liver cells. Animal Cell Technology: from Target to Market 1, 545–547. Kluwer Academic Publishers, London.

    Chapter  Google Scholar 

  42. Demarteau, O., Jakob, M., Schafer, D., Heberer, M., and Martin, I. (2003) Development and validation of a bioreactor for physical stimulation of engineered cartilage. Biorheology 40, 331–336.

    PubMed  CAS  Google Scholar 

  43. Rubin, J., Rubin, C., and Jacobs, C. R. (2006) Molecular pathways mediating mechanical signaling in bone. Gene 367, 1–16.

    Article  PubMed  CAS  Google Scholar 

  44. Rodriguez, A., Cao, Y. L., Ibarra, C., Pap, S., Vacanti, M., Eavey, R. D., and Vacanti, C. A. (1999) Characteristics of cartilage engineered from human pediatric auricular cartilage. Plast. Reconstr. Surg. 103, 1111–1119.

    Article  PubMed  CAS  Google Scholar 

  45. O’Connell, P. (2002) Pancreatic islet xenotransplantation. Xenotransplantation 9, 367–371.

    Article  PubMed  Google Scholar 

  46. O’Connor, S. M., Stenger, D. A., Shaffer, K. M., Maric, D., Barker, J. L., and Ma, W. (2000) Primary neural precursor cell expansion, differentiation and cytosolic Ca2+ response in three-dimensional collagen gel. J. Neurosci. Methods 102, 187–195.

    Article  PubMed  Google Scholar 

  47. Gage, F. H. (2000) Mammalian neural stem cells. Science 287, 1433–1438.

    Article  PubMed  CAS  Google Scholar 

  48. Teng, Y. D., Lavik, E. B., Qu, X. L., Park, K. I., Ourednik, J., Zurakowski, D., Langer, R., and Snyder, E. Y. (2002) Functional recovery following traumatic spinal cord injury mediated by a unique polymer scaffold seeded with neural stem cells. Proc Natl. Acad. Sci. 99, 3024–3029.

    Article  PubMed  CAS  Google Scholar 

  49. Levenberg, S., Golub, J. S., Amit, M., Itskovitz-Eldor, J., and Langer, R. (2002) Endothelial cells derived from human embryonic stem cells. Proc Natl. Acad. Sci. 99, 4391–4396.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Materials Science and Engineering, Kroto Research Institute, University of Sheffield, Sheffield, UK

    John W. Haycock

Authors
  1. John W. Haycock
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to John W. Haycock .

Editor information

Editors and Affiliations

  1. Kroto Research Institute, Department of Engineering Materials, University of Sheffield, Broad Lane, Sheffield, S3 7HW, United Kingdom

    John W. Haycock

Rights and permissions

Reprints and permissions

Copyright information

© 2011 Humana Press

About this protocol

Cite this protocol

Haycock, J.W. (2011). 3D Cell Culture: A Review of Current Approaches and Techniques. In: Haycock, J. (eds) 3D Cell Culture. Methods in Molecular Biology, vol 695. Humana Press. https://doi.org/10.1007/978-1-60761-984-0_1

Download citation

  • DOI: https://doi.org/10.1007/978-1-60761-984-0_1

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60761-983-3

  • Online ISBN: 978-1-60761-984-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation