Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Fabrication of functional three-dimensional tissues by stacking cell sheets in vitro

Nature Protocols volume 7, pages 850–858 (2012)Cite this article

Subjects

This article has been updated

Abstract

The fabrication of 3D tissues retaining the original functions of tissues/organs in vitro is crucial for optimal tissue engineering and regenerative medicine. The fabrication of 3D tissues also contributes to the establishment of in vitro tissue/organ models for drug screening. Our laboratory has developed a fabrication system for functional 3D tissues by stacking cell sheets of confluent cultured cells detached from a temperature-responsive culture dish. Here we describe the protocols for the fabrication of 3D tissues by cell sheet engineering. Three-dimensional cardiac tissues fabricated by stacking cardiac cell sheets pulsate spontaneously, synchronously and macroscopically. Via this protocol, it is also possible to fabricate other tissues, such as 3D tissue including capillary-like prevascular networks, from endothelial cells sandwiched between layered cell sheets. Cell sheet stacking technology promises to provide in vitro tissue/organ models and more effective therapies for curing tissue/organ failures.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: The fabrication of 3D cell-dense tissues by cell sheet engineering.
Figure 2: Layering of cell sheets using a simple pipetting method.
Figure 3: Preparation of a hydrogel-coated, plunger-like manipulator and cell sheet manipulation.
Figure 4: The electrophysiological analyses of cardiac cell sheets.
Figure 5: The fabrication of a multilayered tissue and a prevascularized tissue using a plunger-like manipulator.

Similar content being viewed by others

Change history

  • 03 May 2013

     In the version of this article initially published, the amount of gelatin powder to be added to Hank's balanced salt solution to give a 10-ml total volume when fabricating a gel was given as 0.75 mg. It should be 0.75 g. The error has been corrected in the HTML and PDF versions of the article.

References

  1. Atala, A. et al. Principles of Regenerative Medicine 2nd. ed. (Academic Press,, 2011).

  2. Menasche, P. et al. Myoblast transplantation for heart failure. Lancet 357, 279–280 (2001).

    Article  CAS  PubMed  Google Scholar 

  3. Zhang, M. et al. Cardiomyocyte grafting for cardiac repair: graft cell death and anti-death strategies. J. Mol. Cell Cardiol. 33, 907–921 (2001).

    Article  CAS  PubMed  Google Scholar 

  4. Suzuki, K. et al. Dynamics and mediators of acute graft attrition after myoblast transplantation to the heart. FASEB J. 18, 1153–1155 (2004).

    Article  CAS  PubMed  Google Scholar 

  5. Hofmann, M. et al. Monitoring of bone marrow cell homing into the infarcted human myocardium. Circulation 111, 2198–2202 (2005).

    Article  PubMed  Google Scholar 

  6. Langer, R. & Vacanti, J.P. Tissue engineering. Science 260, 920–926 (1993).

    Article  CAS  PubMed  Google Scholar 

  7. Li, R.K. et al. Survival and function of bioengineered cardiac grafts. Circulation 100, II63–II69 (1999).

    Article  CAS  PubMed  Google Scholar 

  8. Leor, J. et al. Bioengineered cardiac grafts: a new approach to repair the infarcted myocardium? Circulation 102, III56–III61 (2000).

    Article  CAS  PubMed  Google Scholar 

  9. Zimmermann, W.H. et al. Engineered heart tissue grafts improve systolic and diastolic function in infarcted rat hearts. Nat. Med. 12, 452–458 (2006).

    Article  CAS  PubMed  Google Scholar 

  10. Chachques, J.C. et al. Myocardial assistance by grafting a new bioartificial upgraded myocardium (MAGNUM clinical trial): one year follow-up. Cell Transplant. 16, 927–934 (2007).

    Article  PubMed  Google Scholar 

  11. Shinoka, T. & Breuer, C. Tissue-engineered blood vessels in pediatric cardiac surgery. J. Biol. Med. 81, 161–166 (2008).

    Google Scholar 

  12. Iwasa, J. et al. Clinical application of scaffolds for cartilage tissue engineering. Knee Surg. Sports Traumatol. Arthrosc. 17, 561–577 (2009).

    Article  PubMed  Google Scholar 

  13. Lee, K. et al. Cell therapy for bone regeneration—bench to bedside. J. Biomed. Mater. Res. B. Appl. Biomater. 89, 252–263 (2009).

    Article  PubMed  CAS  Google Scholar 

  14. Yamada, N. et al. Thermo-responsive polymeric surface: control of attachment and detachment of cultured cells. Makromol. Chem. Rapid Commun. 11, 571–576 (1990).

    Article  CAS  Google Scholar 

  15. Okano, T. et al. A novel recovery system for cultured cells using plasma-treated polystyrene dishes grafted with poly (N-isopropylacrylamide). J. Biomed. Mater. Res. 27, 1243–1251 (1993).

    Article  CAS  PubMed  Google Scholar 

  16. Yamato, M. et al. Thermo-responsive culture dishes allow the intact harvest of multilayered keratinocyte sheets without dispase by reducing temperature. Tissue Eng. 7, 473–480 (2001).

    Article  CAS  PubMed  Google Scholar 

  17. Matsuda, N. et al. Tissue engineering based on cell sheet technology. Adv. Mater. 19, 3089–3099 (2007).

    Article  CAS  Google Scholar 

  18. Yang, J. et al. Reconstruction of functional tissues with cell sheet engineering. Biomaterials 28, 5033–5043 (2007).

    Article  CAS  PubMed  Google Scholar 

  19. Masuda, S. et al. Cell sheet engineering for heart tissue repair. Adv. Drug. Deliv. Rev. 60, 277–285 (2008).

    Article  CAS  PubMed  Google Scholar 

  20. Hannachi, I.E. et al. Cell sheet engineering: a unique nanotechnology for scaffold-free tissue reconstruction with clinical applications in regenerative medicine. J. Intern. Med. 267, 54–70 (2010).

    Article  CAS  Google Scholar 

  21. Kushida, A. et al. Decrease in culture temperature releases monolayer endothelial cell sheets together with deposited fibronectin matrix from temperature-responsive culture surface. J. Biomed. Mater. Res. 45, 355–362 (1999).

    Article  CAS  PubMed  Google Scholar 

  22. Nishida, K. et al. Functional bioengineered corneal epithelial sheet grafts from corneal stem cells expanded ex vivo on a temperature-responsive cell culture surface. Transplantation 77, 379–385 (2004).

    Article  PubMed  Google Scholar 

  23. Ohashi, K. et al. Engineering functional two- and three-dimensional liver systems in vivo using hepatic tissue sheets. Nat. Med. 13, 880–885 (2007).

    Article  CAS  PubMed  Google Scholar 

  24. Sekine, H. et al. Cardiomyocyte bridging between hearts and bioengineered myocardial tissues with mesenchymal transition of mesothelial cells. J. Heart Lung Transplant. 25, 324–332 (2006).

    Article  PubMed  Google Scholar 

  25. Forte, G. et al. Human cardiac progenitor cell grafts as unrestricted source of supernumerary cardiac cells in healthy murine hearts. Stem Cells 29, 2051–2061 (2011).

    Article  PubMed  Google Scholar 

  26. Sekine, H. et al. Cardiac cell sheet transplantation improves damaged heart function via superior cell survival in comparison with dissociated cell injection. Tissue Eng. Part A 17, 2973–2980 (2011).

    Article  CAS  PubMed  Google Scholar 

  27. Shimizu, T. et al. Fabrication of pulsatile cardiac tissue grafts using a novel 3-dimensional cell sheet manipulation technique and temperature-responsive cell culture surfaces. Circ. Res. 90, e40–e48 (2002).

    Article  CAS  PubMed  Google Scholar 

  28. Shimizu, T. et al. Cell sheet engineering for myocardial tissue reconstruction. Biomaterials 24, 2309–2316 (2003).

    Article  CAS  PubMed  Google Scholar 

  29. Shimizu, T. et al. Long-term survival and growth of pulsatile myocardial tissue grafts engineered by the layering of cardiomyocyte sheets. Tissue Eng. 12, 499–507 (2006).

    Article  CAS  PubMed  Google Scholar 

  30. Shimizu, H. et al. Bioengineering of a functional sheet of islet cells for the treatment of diabetes mellitus. Biomaterials 30, 5943–5949 (2009).

    Article  CAS  PubMed  Google Scholar 

  31. Arauchi, A. et al. Tissue-engineered thyroid cell sheet rescued hypothyroidism in rat models after receiving total thyroidectomy comparing with nontransplantation models. Tissue Eng. Part A. 15, 3943–3949 (2009).

    Article  CAS  PubMed  Google Scholar 

  32. Ohki, T. et al. Treatment of oesophageal ulcerations using endoscopic transplantation of tissue-engineered autologous oral mucosal epithelial cell sheets in a canine model. Gut 55, 1704–1710 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Hata, H. et al. Grafted skeletal myoblast sheets attenuate myocardial remodeling in pacing-induced canine heart failure model. J. Thorac. Cardiovasc. Surg. 132, 918–924 (2006).

    Article  PubMed  Google Scholar 

  34. Miyahara, Y. et al. Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction. Nat. Med. 12, 459–465 (2006).

    Article  CAS  PubMed  Google Scholar 

  35. Kanzaki, M. et al. Functional closure of visceral pleural defects by autologous tissue engineered cell sheets. Eur. J. Cardiothorac. Surg. 34, 864–869 (2008).

    Article  PubMed  Google Scholar 

  36. Hida, N. et al. Novel cardiac precursor-like cells from human menstrual blood-derived mesenchymal cells. Stem Cells 26, 1695–1704 (2008).

    Article  CAS  PubMed  Google Scholar 

  37. Matsuura, K. et al. Transplantation of cardiac progenitor cells ameliorates cardiac dysfunction after myocardial infarction in mice. J. Clin. Invest. 119, 2204–2217 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Iwata, T. et al. Periodontal regeneration with multi-layered periodontal ligament-derived cell sheets in a canine model. Biomaterials 30, 2716–2723 (2009).

    Article  CAS  PubMed  Google Scholar 

  39. Miyagawa, S. et al. Impaired myocardium regeneration with skeletal cell sheets--a preclinical trial for tissue-engineered regeneration therapy. Transplantation 90, 364–372 (2010).

    Article  PubMed  Google Scholar 

  40. Nishida, K. et al. Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium. N. Engl. J. Med. 351, 1187–1196 (2004).

    Article  CAS  PubMed  Google Scholar 

  41. Burillon, C. et al. Cultured autologous oral mucosal epithelial cell-sheet (CAOMECS) transplantation for the treatment of corneal limbal epithelial stem cell deficiency. Invest. Ophthalmol. Vis. Sci. 7 (2011) doi: 10.1167/iovs.11-7744.

  42. Memon, I.A. et al. Repair of impaired myocardium by means of implantation of engineered autologous myoblast sheets. J. Thorac. Cardiovasc. Surg. 130, 1333–1341 (2005).

    Article  PubMed  Google Scholar 

  43. Wei, F.L. et al. Vitamin C treatment promotes mesenchymal stem cell sheet formation and tissue regeneration by elevating telomerase activity. J. Cell Physiol. 21 (2011) doi: 10.1002/jcp.24012.

  44. Sekiya, N. et al. Layered implantation of myoblast sheets attenuates adverse cardiac remodeling of the infarcted heart. J. Thorac. Cardiovasc. Surg. 138, 985–993 (2009).

    Article  PubMed  Google Scholar 

  45. Abbott, A. Biology's new dimension. Nature 424, 870 (2003).

    Article  CAS  PubMed  Google Scholar 

  46. Editorial. Goodbye, flat biology? Nature 424, 861 (2003).

  47. Smalley, K.S. et al. Life isn't flat: taking cancer biology to the next dimension. In Vitro Cell Dev. Biol. Anim. 42, 242 (2006).

    Article  CAS  PubMed  Google Scholar 

  48. Tsuda, Y. et al. Cellular control of tissue architectures using a three-dimensional tissue fabrication technique. Biomaterials 28, 4939–4946 (2007).

    Article  CAS  PubMed  Google Scholar 

  49. Asakawa, N. et al. Pre-vascularization of in vitro three-dimensional tissues created by cell sheet engineering. Biomaterials 31, 3903–3909 (2010).

    Article  CAS  PubMed  Google Scholar 

  50. Sasagawa, T. et al. Design of prevascularized three-dimensional cell-dense tissues using a cell sheet stacking manipulation technology. Biomaterials 31, 1646–1654 (2010).

    Article  CAS  PubMed  Google Scholar 

  51. Sekine, H. et al. Endothelial cell coculture within tissue-engineered cardiomyocyte sheets enhances neovascularization and improves cardiac function of ischemic hearts. Circulation 118, S145–S152 (2008).

    Article  CAS  PubMed  Google Scholar 

  52. Haraguchi, Y. et al. Electrical coupling of cardiomyocyte sheets occurs rapidly via functional gap junction formation. Biomaterials 27, 4765–4774 (2006).

    Article  CAS  PubMed  Google Scholar 

  53. Haraguchi, Y. et al. Development of a new assay system for evaluating the permeability of various substances through three-dimensional tissue. Tissue Eng. Part C 16, 685–692 (2010).

    Article  CAS  Google Scholar 

  54. Hannachi, I.E. et al. Fabrication of transferable micropatterned-co-cultured cell sheets with microcontact printing. Biomaterials 30, 5427–5432 (2009).

    Article  CAS  Google Scholar 

  55. Shimizu, T. et al. Polysurgery of cell sheet grafts overcomes diffusion limits to produce thick, vascularized myocardial tissues. FASEB J. 20, 708–710 (2006).

    Article  CAS  PubMed  Google Scholar 

  56. Miyagawa, S. et al. Tissue cardiomyoplasty using bioengineered contractile cardiomyocyte sheets to repair damaged myocardium: their integration with recipient myocardium. Transplantation 80, 1586–1595 (2005).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the Global Center of Excellence Program, Multidisciplinary Education and Research Center for the establishment of Regenerative Medicine (MERCREM); the Innovation Center for Fusion of Advanced Technologies by special coordination funds for promoting science; the High-Tech Research Center Program from the Ministry of Education, Culture, Sports, Science and Technology (MEXST), Japan; and the Japan Society for the Promotion of Science (JSPS) through the ′Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program),′ initiated by the Council for Science and Technology Policy (CSTP).

Author information

Author notes

  1. Yuji Haraguchi and Tatsuya Shimizu: These authors contributed equally to this study and the writing of the manuscript.

Authors and Affiliations

  1. Institute of Advanced Biomedical Engineering and Science, TWIns, Tokyo Women's Medical University, Tokyo, Japan

    Yuji Haraguchi, Tatsuya Shimizu, Tadashi Sasagawa, Hidekazu Sekine, Katsuhisa Sakaguchi, Tetsutaro Kikuchi, Waki Sekine, Sachiko Sekiya, Masayuki Yamato & Teruo Okano

  2. School of Creative Science and Engineering, TWIns, Waseda University, Tokyo, Japan

    Katsuhisa Sakaguchi & Mitsuo Umezu

Authors
  1. Yuji Haraguchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tatsuya Shimizu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tadashi Sasagawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hidekazu Sekine
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Katsuhisa Sakaguchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Tetsutaro Kikuchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Waki Sekine
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Sachiko Sekiya
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Masayuki Yamato
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Mitsuo Umezu
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Teruo Okano
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Y.H. designed and conducted experiments, analyzed data and wrote the paper. T. Shimizu designed and conducted experiments, analyzed data and wrote the paper, and supervised this project. T. Sasagawa, H.S., K.S., T.K., W.S. and S.S. designed and conducted experiments. M.Y., M.U. and T.O. supervised the project.

Corresponding author

Correspondence to Teruo Okano.

Ethics declarations

Competing interests

Tatsuya Shimizu and Yamato Masayuki are consultants for CellSeed, Inc. Tetsutaro Kikuchi is an employee of CellSeed, Inc. Teruo Okano is an investor in CellSeed, Inc. and an investor/developer designated on the patent for temperature-responsive culture surfaces. No other competing financial interests exist.

Supplementary information

Supplementary Video 1

Layering of two mouse myoblast C2C12 cell sheets using a simple pipetting method. The C2C12 cell sheets were fabricated as described in method 4 of the Supplementary Methods. The size of the two culture dishes is 35 mm. (MOV 9567 kb)

Supplementary Video 2

Preparation of a gelatin gel-coated plunger-like manipulator and placement of the manipulator with a plunger-guiding cover onto a temperature-responsive culture dish. Similar procedures are performed when using fibrin gel. (MOV 9457 kb)

Supplementary Video 3

Transplantation of a layered-cardiac cell sheet onto the dorsal subcutaneous tissue. The cell sheet constructs were lifted onto a sterile polyethylene terephthalate (PET) film and transplanted onto the dorsal subcutaneous tissue by sliding it from the PET film using two forceps (straight and warping type). (MOV 2018 kb)

Supplementary Video 4

Detaching of a cardiac cell sheet by low temperature treatment (20 oC). The phase contrast microscopic image was recorded by a digital video recorder through a CCD camera. (MOV 3170 kb)

Supplementary Video 5

Spontaneous, synchronous, and macroscopic beating of a layered cardiac cell sheet in a culture dish and on fibrin gel. These layered cell sheets were macroscopically observed by using a digital video camera. (MOV 3747 kb)

Supplementary Methods

Fabrication of a cardiac cell sheet using neonatal rat cardiac cells; Preparation of a temperature-responsive culture dish; Fabrication and histological observation of prevascularized three-dimensional cell-dense tissues; and Fabrication of a C2C12 cell sheet. (PDF 90 kb)

Supplementary Table 1

Fabrication conditions for several cell sheets. (PDF 35 kb)

Supplementary Figures 1–5

Supplementary Fig. 1 (PDF 717 kb)

Detailed explanation of cell sheet layering using a simple pipetting method. (a) Spread and adhere the first recovered cell sheet onto culture material. (b) To layer the cell sheets, place and spread a second recovered cell sheet onto the dish which will adhere to the first cell sheet. Three-dimensional tissue is fabricated by repeating the same procedures. All culture dishes in the figure are 35-mm.

Supplementary Fig. 2

Detailed explanation of cell sheet layering using a hydrogel-coated plunger-like manipulator. (a) The upper silicon mold with a circular hole is put on the lower silicon mold. (b) The color of the gelatin solution changes to red from yellow by the addition of NaOH. (c) A hydrogel-coated plunger-like manipulator is fabricated by using hydrogel solution, two silicone molds, and a plunger-like manipulator. (d, e) After removing the hydrogel overflow, take out the two silicon molds from the manipulator to expose the hydrogel. (f) Place the hydrogel-coated manipulator with a plunger-guiding cover on a temperature-responsive culture dish (TRCD) containing the confluent cells. (g) After incubation at 20 °C, lift up the manipulator holding a cell sheet. After repeating the same procedures, recover a multi-layered cell sheet on hydrogel by using a scalpel and transfer onto another culture material by using forceps (h) or use directly for transplantation.

Supplementary Fig. 3

The dimensions of the silicon molds and the hole (a), and the plunger-like manipulator with the guiding cover (b). The thicknesses of the silicon molds are approximately 1-2 mm. These dimensions of the manipulators are applied to a 35-mm culture dish and can be scaled up/down depending on the dish size. For example, in the case of a 60-mm dish, the hole size in the silicon mold would be 48-mm.

Supplementary Fig. 4

Recovery of an intact cell sheet by seeding with the optimal cell numbers. (a) With optimal cell numbers an intact cell sheet can be recovered. (b) With less than optimal cell numbers, a broken cell sheet or a cell sheet having many holes is recovered. (c) With sparse number of cells, seeded cells cannot be recovered as an intact cell sheet. All culture dishes are 35 mm.

Supplementary Fig. 5

Transfer a layered cell sheet construct with hydrogel onto another culture material and subsequent in vitro culture of the construct. (a) The cultivation of a layered cell sheet construct on fibrin gel. (b) After re-adhering a layered cell sheet construct onto a culture material surface and removing the gelatin gel, it forms a layered cell sheet construct without any gel.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Haraguchi, Y., Shimizu, T., Sasagawa, T. et al. Fabrication of functional three-dimensional tissues by stacking cell sheets in vitro. Nat Protoc 7, 850–858 (2012). https://doi.org/10.1038/nprot.2012.027

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2012.027

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research