Chinese Science Bulletin

, Volume 58, Issue 35, pp 4349–4356 | Cite as

Regenerative medicine of cornea by cell sheet engineering using temperature-responsive culture surfaces

  • Terumasa Umemoto
  • Masayuki Yamato
  • Kohji Nishida
  • Teruo Okano
Open Access
Review Special Topic 9th World Biomaterials Congress: Innovative Biomaterials and Crossing Frontiers in Biomaterials and Regenerative M


Recently, regenerative medicine has been focused on as next-generation definitive therapies for several diseases or injuries for which there are currently no effective treatments. These therapies have been rapidly developed through the effective fusion between different fields such as stem cell biology and biomaterials. So far, we have proposed “cell sheet engineering” through our core technology that simply applies alterations of the temperature which allows regulation of the attachment or detachment of living cells on the culture surfaces grafted with the temperature-responsive polymer “poly(N-isoproplyacrylamide)”. This technology enables us to construct bioengineered sheet-like tissues, termed “cell sheets”, without the need of using biodegradable scaffolds and protease treatments that are traditionally used. Therefore, our carrier-free cell sheets not only are independent of the issues observed in conventional methods, but also showed further advantages in the reconstruction of the corneal epithelium or endothelium (e.g. improvement of optical transparency and cell-cell interactions to host stroma in reconstructed tissues). Moreover, our approach does not have issues such as immune rejection or limited number of donors, due to the use of cell sheets derived from autologous limbal (in patients with unilateral disease) or oral mucosal epithelial cells (in patients with bilateral disorders). Indeed, we have successfully performed the clinical application for the reconstruction of ocular surfaces through the transplantation of our carrier-free corneal epithelial cell sheets, as evidenced by improved visual acuity as well as long-term maintenance of postoperative health conditions on ocular surfaces in all patients. We have also proposed a novel approach for the reconstruction of the corneal endothelium using corneal endothelial cell sheets by demonstrating its successful application. Thus, our cell sheet engineering technique provides a breakthrough in the field of regenerative medicine applied to the cornea.


cell sheet temperature-responsive carrier-free 


  1. 1.
    Gallico G G, O’Connor N E, Compton C C, et al. Permanent coverage of large burn wounds with autologous cultured human epithelium. N Engl J Med, 1984, 311: 448–451CrossRefGoogle Scholar
  2. 2.
    Gallico G G, O’Connor N E, Compton C C, et al. Cultured epithelial autografts for giant congenital nevi. Plast Reconstr Surg, 1989, 84: 1–9CrossRefGoogle Scholar
  3. 3.
    Phillips T J, Kehinde O, Green H, et al. Treatment of skin ulcers with cultured epidermal allografts. J Am Acad Dermatol, 1989, 21: 191–199CrossRefGoogle Scholar
  4. 4.
    Langer R, Vacanti J P. Tissue engineering. Science, 1993, 260: 920–926CrossRefGoogle Scholar
  5. 5.
    Vacanti J P. Beyond transplantation. Third annual samuel jason mixter lecture. Arch Surg, 1988, 123: 545–549CrossRefGoogle Scholar
  6. 6.
    Vacanti J P, Langer R. Tissue engineering: The design and fabrication of living replacement devices for surgical reconstruction and transplantation. Lancet, 1999, 354(Suppl 1): 132–134Google Scholar
  7. 7.
    Atala A, Bauer S B, Soker S, et al. Tissue-engineered autologous bladders for patients needing cystoplasty. Lancet, 2006, 367: 1241–1246CrossRefGoogle Scholar
  8. 8.
    Poh M, Boyer M, Solan A, et al. Blood vessels engineered from human cells. Lancet, 2005, 365: 2122–2124CrossRefGoogle Scholar
  9. 9.
    Shin’oka T, Imai Y, Ikada Y. Transplantation of a tissue-engineered pulmonary artery. N Engl J Med, 2001, 344: 532–533CrossRefGoogle Scholar
  10. 10.
    Vacanti C A, Bonassar L J, Vacanti M P, et al. Replacement of an avulsed phalanx with tissue-engineered bone. N Engl J Med, 2001, 344: 1511–1514CrossRefGoogle Scholar
  11. 11.
    Pellegrini G, Traverso C E, Franzi A T, et al. Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium. Lancet, 1997, 349: 990–993CrossRefGoogle Scholar
  12. 12.
    Buck R C. Measurement of centripetal migration of normal corneal epithelial cells in the mouse. Invest Ophthalmol Vis Sci, 1985, 26: 1296–1299Google Scholar
  13. 13.
    Kinoshita S, Friend J, Thoft R A. Sex chromatin of donor corneal epithelium in rabbits. Invest Ophthalmol Vis Sci, 1981, 21: 434–441Google Scholar
  14. 14.
    Thoft R A, Friend J. The X, Y, Z hypothesis of corneal epithelial maintenance. Invest Ophthalmol Vis Sci, 1983, 24: 1442–1443Google Scholar
  15. 15.
    Cotsarelis G, Cheng S Z, Dong G, et al. Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: Implications on epithelial stem cells. Cell, 1989, 57: 201–209CrossRefGoogle Scholar
  16. 16.
    Schermer A, Galvin S, Sun T T. Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells. J Cell Biol, 1986, 103: 49–62CrossRefGoogle Scholar
  17. 17.
    Nishida K, Yamato M, Hayashida Y, et al. Functional bioengineered corneal epithelial sheet grafts from corneal stem cells expanded ex vivo on a temperature-responsive cell culture surface. Transplantation, 2004, 77: 379–385CrossRefGoogle Scholar
  18. 18.
    Yamato M, Utsumi M, Kushida A, et al. Thermo-responsive culture dishes allow the intact harvest of multilayered keratinocyte sheets without dispase by reducing temperature. Tissue Eng, 2001, 7: 473–480CrossRefGoogle Scholar
  19. 19.
    Han B, Schwab I R, Madsen T K, et al. A fibrin-based bioengineered ocular surface with human corneal epithelial stem cells. Cornea, 2002, 21: 505–510CrossRefGoogle Scholar
  20. 20.
    Rama P, Bonini S, Lambiase A, et al. Autologous fibrin-cultured limbal stem cells permanently restore the corneal surface of patients with total limbal stem cell deficiency. Transplantation, 2001, 72: 1478–1485CrossRefGoogle Scholar
  21. 21.
    Schwab I R. Cultured corneal epithelia for ocular surface disease. Trans Am Ophthalmol Soc, 1999, 97: 891–986Google Scholar
  22. 22.
    Tsai R J, Li L M, Chen J K. Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells. N Engl J Med, 2000, 343: 86–93CrossRefGoogle Scholar
  23. 23.
    Okano T, Yamada N, Okuhara M, et al. Mechanism of cell detachment from temperature-modulated, hydrophilic-hydrophobic polymer surfaces. Biomaterials, 1995, 16: 297–303CrossRefGoogle Scholar
  24. 24.
    Okano T, Yamada N, Sakai H, et al. A novel recovery system for cultured cells using plasma-treated polystyrene dishes grafted with poly(N-isopropylacrylamide). J Biomed Mater Res, 1993, 27: 1243–1251CrossRefGoogle Scholar
  25. 25.
    Kushida A, Yamato M, Konno C, et al. Decrease in culture temperature releases monolayer endothelial cell sheets together with deposited fibronectin matrix from temperature-responsive culture surfaces. J Biomed Mater Res, 1999, 45: 355–362CrossRefGoogle Scholar
  26. 26.
    Kushida A, Yamato M, Isoi Y, et al. A noninvasive transfer system for polarized renal tubule epithelial cell sheets using temperature-responsive culture dishes. Eur Cell Mater, 2005, 10: 23–30Google Scholar
  27. 27.
    Kushida A, Yamato M, Kikuchi A, et al. Two-dimensional manipulation of differentiated Madin-Darby canine kidney (MDCK) cell sheets: The noninvasive harvest from temperature-responsive culture dishes and transfer to other surfaces. J Biomed Mater Res, 2001, 54: 37–46CrossRefGoogle Scholar
  28. 28.
    Akizuki T, Oda S, Komaki M, et al. Application of periodontal ligament cell sheet for periodontal regeneration: A pilot study in beagle dogs. J Periodontal Res, 2005, 40: 245–251CrossRefGoogle Scholar
  29. 29.
    Hasegawa M, Yamato M, Kikuchi A, et al. Human periodontal ligament cell sheets can regenerate periodontal ligament tissue in an athymic rat model. Tissue Eng, 2005, 11: 469–478CrossRefGoogle Scholar
  30. 30.
    Yaji N, Yamato M, Yang J, et al. Transplantation of tissue-engineered retinal pigment epithelial cell sheets in a rabbit model. Biomaterials, 2009, 3: 797–803CrossRefGoogle Scholar
  31. 31.
    Arauchi A, Shimizu T, Yamato M, et al. Tissue-engineered thyroid cell sheet rescued hypothyroidism in rat models after receiving total thyroidectomy comparing with nontransplantation models. Tissue Eng Part A, 2009, 15: 3943–3949CrossRefGoogle Scholar
  32. 32.
    Uchiyama H, Yamato M, Sasaki R, et al. In vivo 3D analysis with micro-computed tomography of rat calvaria bone regeneration using periosteal cell sheets fabricated on temperature-responsive culture dishes. J Tissue Eng Regen Med, 2011, 5: 483–490CrossRefGoogle Scholar
  33. 33.
    Shimizu T, Yamato M, Akutsu T, et al. Electrically communicating three-dimensional cardiac tissue mimic fabricated by layered cultured cardiomyocyte sheets. J Biomed Mater Res, 2002, 60: 110–117CrossRefGoogle Scholar
  34. 34.
    Shimizu T, Yamato M, Kikuchi A, et al. Two-dimensional manipulation of cardiac myocyte sheets utilizing temperature-responsive culture dishes augments the pulsatile amplitude. Tissue Eng, 2001, 7: 141–151CrossRefGoogle Scholar
  35. 35.
    Ohki T, Yamato M, Ota M, et al. Prevention of esophageal stricture after endoscopic submucosal dissection using tissue-engineered cell sheets. Gastroenterology, 2012, 143: 582–588CrossRefGoogle Scholar
  36. 36.
    Nishida K, Yamato M, Hayashida Y, et al. Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium. N Engl J Med, 2004, 351: 1187–1196CrossRefGoogle Scholar
  37. 37.
    Haraguchi Y, Shimizu T, Sasagawa T, et al. Fabrication of functional three-dimensional tissues by stacking cell sheets in vitro. Nat Protoc, 2012, 7: 850–858CrossRefGoogle Scholar
  38. 38.
    Shimizu T, Sekine H, Yang J, et al. Polysurgery of cell sheet grafts overcomes diffusion limits to produce thick, vascularized myocardial tissues. Faseb J, 2006, 20: 708–710Google Scholar
  39. 39.
    Shimizu T, Yamato M, Isoi Y, 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, 2002, 90: e40CrossRefGoogle Scholar
  40. 40.
    Yamada N, Okano T, Sakai H, et al. Thermo-responsive polymeric surfaces; control of attachment and detachment of cultured cells. Makromol Chem Rapid Commun, 1990, 11: 571–576CrossRefGoogle Scholar
  41. 41.
    Pan Y V, Wesley R A, Luginbuhl R, et al. Plasma polymerized N-isopropylacrylamide: Synthesis and characterization of a smart thermally responsive coating. Biomacromolecules, 2001, 2: 32–36CrossRefGoogle Scholar
  42. 42.
    Nagase K, Watanabe M, Kikuchi A, et al. Thermo-responsive polymer brushes as intelligent biointerfaces: Preparation via ATRP and characterization. Macromol Biosci, 2011, 11: 400–409CrossRefGoogle Scholar
  43. 43.
    Takahashi H, Nakayama M, Yamato M, et al. Controlled chain length and graft density of thermoresponsive polymer brushes for optimizing cell sheet harvest. Biomacromolecules, 2010, 11: 1991–1999CrossRefGoogle Scholar
  44. 44.
    Mizutani A, Kikuchi A, Yamato M, et al. Preparation of thermoresponsive polymer brush surfaces and their interaction with cells. Biomaterials, 2008, 29: 2073–2081CrossRefGoogle Scholar
  45. 45.
    Li L, Zhu Y, Li B, et al. Fabrication of thermoresponsive polymer gradients for study of cell adhesion and detachment. Langmuir, 2008, 24: 13632–13639CrossRefGoogle Scholar
  46. 46.
    Xu F J, Zhong S P, Yung L Y, et al. Surface-active and stimuli-responsive polymer—Si(100) hybrids from surface-initiated atom transfer radical polymerization for control of cell adhesion. Biomacromolecules, 2004, 5: 2392–2403CrossRefGoogle Scholar
  47. 47.
    Nakayama M, Yamada N, Kumashiro Y, et al. Thermoresponsive poly(N-isopropylacrylamide)-based block copolymer coating for optimizing cell sheet fabrication. Macromol Biosci, 2012, 12: 751–760CrossRefGoogle Scholar
  48. 48.
    Ebara M, Yamato M, Aoyagi T, et al. The effect of extensible PEG tethers on shielding between grafted thermo-responsive polymer chains and integrin-RGD binding. Biomaterials, 2008, 29: 3650–3655CrossRefGoogle Scholar
  49. 49.
    Takahashi H, Nakayama M, Shimizu T, et al. Anisotropic cell sheets for constructing three-dimensional tissue with well-organized cell orientation. Biomaterials, 2011, 32: 8830–8838CrossRefGoogle Scholar
  50. 50.
    Takahashi H, Nakayama M, Itoga K, et al. Micropatterned thermoresponsive polymer brush surfaces for fabricating cell sheets with well-controlled orientational structures. Biomacromolecules, 2011, 12: 1414–1418CrossRefGoogle Scholar
  51. 51.
    Muraoka M, Shimizu T, Itoga K, et al. Control of the formation of vascular networks in 3D tissue engineered constructs. Biomaterials, 2013, 34: 696–703CrossRefGoogle Scholar
  52. 52.
    Tsuda Y, Shimizu T, Yamato M, et al. Cellular control of tissue architectures using a three-dimensional tissue fabrication technique. Biomaterials, 2007, 28: 4939–4946CrossRefGoogle Scholar
  53. 53.
    Kenyon K R, Tseng S C. Limbal autograft transplantation for ocular surface disorders. Ophthalmology, 1989, 96: 709–722CrossRefGoogle Scholar
  54. 54.
    Tsubota K, Satake Y, Kaido M, et al. Treatment of severe ocular-surface disorders with corneal epithelial stem-cell transplantation. N Engl J Med. 1999, 340: 1697–1703CrossRefGoogle Scholar
  55. 55.
    Nakamura T, Endo K, Cooper L J, et al. The successful culture and autologous transplantation of rabbit oral mucosal epithelial cells on amniotic membrane. Invest Ophthalmol Vis Sci, 2003, 44: 106–116CrossRefGoogle Scholar
  56. 56.
    Hayashida Y, Nishida K, Yamato M, et al. Ocular surface reconstruction using autologous rabbit oral mucosal epithelial sheets fabricated ex vivo on a temperature-responsive culture surface. Invest Ophthalmol Vis Sci, 2005, 46: 1632–1639CrossRefGoogle Scholar
  57. 57.
    Takagi R, Yamato M, Murakami D, et al. Preparation of keratinocyte culture medium for the clinical applications of regenerative medicine. J Tissue Eng Regen Med, 2011, 5: 63–73CrossRefGoogle Scholar
  58. 58.
    Murakami D, Yamato M, Nishida K, et al. The effect of micropores in the surface of temperature-responsive culture inserts on the fabrication of transplantable canine oral mucosal epithelial cell sheets. Biomaterials, 2006, 27: 5518–5523CrossRefGoogle Scholar
  59. 59.
    Murakami D, Yamato M, Nishida K, et al. Fabrication of transplantable human oral mucosal epithelial cell sheets using temperature-responsive culture inserts without feeder layer cells. J Artif Organs, 2006, 9: 185–191CrossRefGoogle Scholar
  60. 60.
    Takagi R, Yamato M, Murakami D, et al. Fabrication and validation of autologous human oral mucosal epithelial cell sheets to prevent stenosis after esophageal endoscopic submucosal dissection. Pathobiology, 2011, 78: 311–319CrossRefGoogle Scholar
  61. 61.
    Hayashi R, Yamato M, Takayanagi H, et al. Validation system of tissue-engineered epithelial cell sheets for corneal regenerative medicine. Tissue Eng Part C Methods, 2010, 16: 553–560CrossRefGoogle Scholar
  62. 62.
    Watanabe K, Yamato M, Hayashida Y, et al. Development of transplantable genetically modified corneal epithelial cell sheets for gene therapy. Biomaterials, 2007, 28: 745–749CrossRefGoogle Scholar
  63. 63.
    Kondo M, Yamato M, Takagi R, et al. The regulation of epithelial cell proliferation and growth by IL-1 receptor antagonist. Biomaterials, 2013, 34: 121–129CrossRefGoogle Scholar
  64. 64.
    Takagi R, Yamato M, Kushida A, et al. Profiling of extracellular matrix and cadherin family gene expression in mouse feeder layer cells: Type VI collagen is a candidate molecule inducing the colony formation of epithelial cells. Tissue Eng Part A, 2012, 18: 2539–2548CrossRefGoogle Scholar
  65. 65.
    Obokata H, Yamato M, Tsuneda S, et al. Reproducible subcutaneous transplantation of cell sheets into recipient mice. Nat Protoc, 2011, 6: 1053–1059CrossRefGoogle Scholar
  66. 66.
    Sugiyama H, Maeda K, Yamato M, et al. Human adipose tissue-derived mesenchymal stem cells as a novel feeder layer for epithelial cells. J Tissue Eng Regen Med, 2008, 2: 445–449CrossRefGoogle Scholar
  67. 67.
    Umemoto T, Yamato M, Nishida K, et al. Limbal epithelial side-population cells have stem cell-like properties, including quiescent state. Stem Cells, 2006, 24: 86–94CrossRefGoogle Scholar
  68. 68.
    Dikstein S, Maurice D M. The active control of corneal hydration. Isr J Med Sci, 1972, 8: 1523–1528Google Scholar
  69. 69.
    Murphy C, Alvarado J, Juster R, et al. Prenatal and postnatal cellularity of the human corneal endothelium. A quantitative histologic study. Invest Ophthalmol Vis Sci, 1984, 25: 312–322Google Scholar
  70. 70.
    Svedbergh B, Bill A. Scanning electron microscopic studies of the corneal endothelium in man and monkeys. Acta Ophthalmol, 1972, 50: 321–336CrossRefGoogle Scholar
  71. 71.
    Kaufman H B B, McDonald M. The Cornea. 2nd ed. Woburn, MA: Butterworth-Heinemann, 1998Google Scholar
  72. 72.
    Egan C A, Savre-Train I, Shay J W, et al. Analysis of telomere lengths in human corneal endothelial cells from donors of different ages. Invest Ophthalmol Vis Sci, 1998, 39: 648–653Google Scholar
  73. 73.
    Senoo T, Joyce N C. Cell cycle kinetics in corneal endothelium from old and young donors. Invest Ophthalmol Vis Sci, 2000, 41: 660–667Google Scholar
  74. 74.
    Wilson S E, Weng J, Blair S, et al. Expression of E6/E7 or SV40 large T antigen-coding oncogenes in human corneal endothelial cells indicates regulated high-proliferative capacity. Invest Ophthalmol Vis Sci, 1995, 36: 32–40Google Scholar
  75. 75.
    Sumide T, Nishida K, Yamato M, et al. Functional human corneal endothelial cell sheets harvested from temperature-responsive culture surfaces. Faseb J, 2006, 20: 392–394Google Scholar
  76. 76.
    Ide T, Nishida K, Yamato M, et al. Structural characterization of bioengineered human corneal endothelial cell sheets fabricated on temperature-responsive culture dishes. Biomaterials, 2006, 27: 607–614CrossRefGoogle Scholar

Copyright information

© The Author(s) 2013

Authors and Affiliations

  • Terumasa Umemoto
    • 1
  • Masayuki Yamato
    • 1
  • Kohji Nishida
    • 2
  • Teruo Okano
    • 1
  1. 1.Institute of Advanced Biomedical Engineering and ScienceTokyo Women’s Medical UniversityTokyoJapan
  2. 2.Department of OphthalmologyOsaka University Medical SchoolOsakaJapan

Personalised recommendations