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Vol. 78, No. 6, 2011
Issue release date: November 2011
Section title: Paper
Pathobiology 2011;78:311–319
(DOI:10.1159/000322575)

Fabrication and Validation of Autologous Human Oral Mucosal Epithelial Cell Sheets to Prevent Stenosis after Esophageal Endoscopic Submucosal Dissection

Takagi R.a · Yamato M.a · Murakami D.a, d · Kondo M.a, d · Ohki T.a, b · Sasaki R.a, c · Nishida K.e · Namiki H.d · Yamamoto M.b · Okano T.a
aInstitute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University and Global Center of Excellence, bDepartment of Surgery, Institute of Gastroenterology, and cDepartment of Oral and Maxillofacial Surgery, Tokyo Women’s Medical University, and dGraduate School of Science and Engineering, Waseda University, Tokyo, and eDepartment of Ophthalmology, Osaka University Medical School, Osaka, Japan
email Corresponding Author

Abstract

Objectives: Human oral mucosal epithelial cells derived from 7 healthy volunteer donors were cultured in a clean room in a cell-processing center (CPC) according to good manufacturing practice guidelines. Cell culture and fabricated transplantable epithelial cell sheets were validated for treating ulcers after endoscopic mucosal dissection. Methods: The clonal growth and morphology of the human oral mucosal epithelial cells seeded on temperature-responsive surfaces were observed. During the cultivation, sterilization tests were performed to validate the environment in the CPC. To validate the purity and morphology of fabricated epithelial cell sheets, cell sheets harvested from temperature-responsive surfaces by temperature reduction were examined by histology and flow cytometry. Results: Human oral mucosal epithelial cells were successfully cultured and harvested as continuous cell sheets from temperature-responsive culture inserts without any animal-derived materials. During the cultivations, the sterile environment in the CPC was confirmed. The results of histological and flow cytometry analysis showed the high reproducibility of stratification and the purity of the fabricated human oral mucosal epithelial cell sheets. Conclusions: The method for fabricating epithelial cell sheets shown in this study was suitable for the validation for clinical trials and suggested usability of the fabricated cell sheets.

© 2011 S. Karger AG, Basel


  

Key Words

  • Cell culture
  • Human oral mucosal epithelial cells
  • Esophageal squamous cell carcinoma
  • Flow cytometry

References

  1. Gallico GG 3rd, O’Connor NE, Compton CC, Kehinde O, Green H: Permanent coverage of large burn wounds with autologous cultured human epithelium. N Engl J Med 1984;311:448–451.
  2. Phillips TJ, Kehinde O, Green H, Gilchrest BA: Treatment of skin ulcers with cultured epidermal allografts. J Am Acad Dermatol 1989;21:191–199.
  3. Gallico GG 3rd, O’Connor NE, Compton CC, Remensnyder JP, Kehinde O, Green H: Cultured epithelial autografts for giant congenital nevi. Plast Reconstr Surg 1989;84:1–9.
  4. Rheinwald JG, Green H: Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 1975;6:331–343.
  5. Mathor MB, Ferrari G, Dellambra E, Cilli M, Mavilio F, Cancedda R, De Luca M: Clonal analysis of stably transduced human epidermal stem cells in culture. Proc Natl Acad Sci USA 1996;93:10371–10376.
  6. de Luca M, Albanese E, Megna M, Cancedda R, Mangiante PE, Cadoni A, Franzi AT: Evidence that human oral epithelium reconstituted in vitro and transplanted onto patients with defects in the oral mucosa retains properties of the original donor site. Transplantation 1990;50:454–459.
  7. Ueda M, Hata K, Horie K, Torii S: The potential of oral mucosal cells for cultured epithelium: a preliminary report. Ann Plast Surg 1995;35:498–504.
  8. Nishida K, Yamato M, Hayashida Y, Watanabe K, Yamamoto K, Adachi E, Nagai S, Kikuchi A, Maeda N, Watanabe H, Okano T, Tano Y: Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium. N Engl J Med 2004;351:1187–1196.
  9. Martin MJ, Muotri A, Gage F, Varki A: Human embryonic stem cells express an immunogenic nonhuman sialic acid. Nat Med 2005;11:228–232.
  10. Murakami D, Yamato M, Nishida K, Ohki T, Takagi R, Yang J, Namiki H, Okano T: 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;27:5518–5523.
  11. Murakami D, Yamato M, Nishida K, Ohki T, Takagi R, Yang J, Namiki H, Okano T: Fabrication of transplantable human oral mucosal epithelial cell sheets using temperature-responsive culture inserts without feeder layer cells. J Artif Organs 2006;9:185–191.
  12. Yang J, Yamato M, Kohno C, Nishimoto A, Sekine H, Fukai F, Okano T: Cell sheet engineering: recreating tissues without biodegradable scaffolds. Biomaterials 2005;26:6415–6422.
  13. Yamada N, Okano T, Sakai H, Karikusa F, Sawasaki Y, Sakurai Y: Thermo-responsive polymeric surfaces; control of attachment and detachment of cultured cells. Makromol Chem Rapid Commun 1991;11:571–576.

    External Resources

  14. Ohki T, Yamato M, Murakami D, Takagi R, Yang J, Namiki H, Okano T, Takasaki K: Treatment of oesophageal ulcerations using endoscopic transplantation of tissue-engineered autologous oral mucosal epithelial cell sheets in a canine model. Gut 2006;55:1704–1710.
  15. Hirao M, Masuda K, Asanuma T, Naka H, Noda K, Matsuura K, Yamaguchi O, Ueda N: Endoscopic resection of early gastric cancer and other tumors with local injection of hypertonic saline-epinephrine. Gastrointest Endosc 1988;34:264–269.
  16. Gotoda T, Kondo H, Ono H, Saito Y, Yamaguchi H, Saito D, Yokota T: A new endoscopic mucosal resection procedure using an insulation-tipped electrosurgical knife for rectal flat lesions: report of two cases. Gastrointest Endosc 1999;50:560–563.
  17. Oyama T, Tomori A, Hotta K, Morita S, Kominato K, Tanaka M, Miyata Y: Endoscopic submucosal dissection of early esophageal cancer. Clin Gastroenterol Hepatol 2005;3:S67–70.
  18. Gotoda T, Yamamoto H, Soetikno RM: Endoscopic submucosal dissection of early gastric cancer. J Gastroenterol 2006;41:929–942.
  19. Katada C, Muto M, Manabe T, Boku N, Ohtsu A, Yoshida S: Esophageal stenosis after endoscopic mucosal resection of superficial esophageal lesions. Gastrointest Endosc 2003;57:165–169.
  20. Takagi R, Yamato M, Murakami D, Kondo M, Yang J, Ohki T, Nishida K, Kohno C, Okano T: Preparation of keratinocyte culture medium for the clinical applications of regenerative medicine. J Tissue Eng Regen Med 2011;5:e63–73.
  21. Takagi R, Murakami D, Kondo M, Ohki T, Sasaki R, Mizutani M, Yamato M, Nishida K, Namiki H, Yamamoto M, Okano T: Fabrication of human oral mucosal epithelial cell sheets for treatment of esophageal ulceration by endoscopic submucosal dissection. Gastrointest Endosc 2010;72:1253–1259.

  

Author Contacts

Masayuki Yamato, PhD
Institute of Advanced Biomedical Engineering and Science Tokyo Women’s Medical University, TWIns, 8-1 Kawada-cho, Shinjuku-ku
Tokyo 162-8666 (Japan)
Tel. +81 3 3353 8112, ext. 66211, E-Mail myamato@abmes.twmu.ac.jp

  

Article Information

Published online: November 18, 2011
Number of Print Pages : 9
Number of Figures : 5, Number of Tables : 3, Number of References : 21

  

Publication Details

Pathobiology (Exploring the basis of disease)

Vol. 78, No. 6, Year 2011 (Cover Date: November 2011)

Journal Editor: Borisch B. (Geneva), Yasui W. (Hiroshima)
ISSN: 1015-2008 (Print), eISSN: 1423-0291 (Online)

For additional information: http://www.karger.com/PAT


Copyright / Drug Dosage / Disclaimer

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

Abstract

Objectives: Human oral mucosal epithelial cells derived from 7 healthy volunteer donors were cultured in a clean room in a cell-processing center (CPC) according to good manufacturing practice guidelines. Cell culture and fabricated transplantable epithelial cell sheets were validated for treating ulcers after endoscopic mucosal dissection. Methods: The clonal growth and morphology of the human oral mucosal epithelial cells seeded on temperature-responsive surfaces were observed. During the cultivation, sterilization tests were performed to validate the environment in the CPC. To validate the purity and morphology of fabricated epithelial cell sheets, cell sheets harvested from temperature-responsive surfaces by temperature reduction were examined by histology and flow cytometry. Results: Human oral mucosal epithelial cells were successfully cultured and harvested as continuous cell sheets from temperature-responsive culture inserts without any animal-derived materials. During the cultivations, the sterile environment in the CPC was confirmed. The results of histological and flow cytometry analysis showed the high reproducibility of stratification and the purity of the fabricated human oral mucosal epithelial cell sheets. Conclusions: The method for fabricating epithelial cell sheets shown in this study was suitable for the validation for clinical trials and suggested usability of the fabricated cell sheets.

© 2011 S. Karger AG, Basel


  

Author Contacts

Masayuki Yamato, PhD
Institute of Advanced Biomedical Engineering and Science Tokyo Women’s Medical University, TWIns, 8-1 Kawada-cho, Shinjuku-ku
Tokyo 162-8666 (Japan)
Tel. +81 3 3353 8112, ext. 66211, E-Mail myamato@abmes.twmu.ac.jp

  

Article Information

Published online: November 18, 2011
Number of Print Pages : 9
Number of Figures : 5, Number of Tables : 3, Number of References : 21

  

Publication Details

Pathobiology (Exploring the basis of disease)

Vol. 78, No. 6, Year 2011 (Cover Date: November 2011)

Journal Editor: Borisch B. (Geneva), Yasui W. (Hiroshima)
ISSN: 1015-2008 (Print), eISSN: 1423-0291 (Online)

For additional information: http://www.karger.com/PAT


Article / Publication Details

First-Page Preview
Abstract of Paper

Published online: 11/18/2011
Issue release date: November 2011

Number of Print Pages: 9
Number of Figures: 5
Number of Tables: 3

ISSN: 1015-2008 (Print)
eISSN: 1423-0291 (Online)

For additional information: http://www.karger.com/PAT


Copyright / Drug Dosage

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

References

  1. Gallico GG 3rd, O’Connor NE, Compton CC, Kehinde O, Green H: Permanent coverage of large burn wounds with autologous cultured human epithelium. N Engl J Med 1984;311:448–451.
  2. Phillips TJ, Kehinde O, Green H, Gilchrest BA: Treatment of skin ulcers with cultured epidermal allografts. J Am Acad Dermatol 1989;21:191–199.
  3. Gallico GG 3rd, O’Connor NE, Compton CC, Remensnyder JP, Kehinde O, Green H: Cultured epithelial autografts for giant congenital nevi. Plast Reconstr Surg 1989;84:1–9.
  4. Rheinwald JG, Green H: Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 1975;6:331–343.
  5. Mathor MB, Ferrari G, Dellambra E, Cilli M, Mavilio F, Cancedda R, De Luca M: Clonal analysis of stably transduced human epidermal stem cells in culture. Proc Natl Acad Sci USA 1996;93:10371–10376.
  6. de Luca M, Albanese E, Megna M, Cancedda R, Mangiante PE, Cadoni A, Franzi AT: Evidence that human oral epithelium reconstituted in vitro and transplanted onto patients with defects in the oral mucosa retains properties of the original donor site. Transplantation 1990;50:454–459.
  7. Ueda M, Hata K, Horie K, Torii S: The potential of oral mucosal cells for cultured epithelium: a preliminary report. Ann Plast Surg 1995;35:498–504.
  8. Nishida K, Yamato M, Hayashida Y, Watanabe K, Yamamoto K, Adachi E, Nagai S, Kikuchi A, Maeda N, Watanabe H, Okano T, Tano Y: Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium. N Engl J Med 2004;351:1187–1196.
  9. Martin MJ, Muotri A, Gage F, Varki A: Human embryonic stem cells express an immunogenic nonhuman sialic acid. Nat Med 2005;11:228–232.
  10. Murakami D, Yamato M, Nishida K, Ohki T, Takagi R, Yang J, Namiki H, Okano T: 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;27:5518–5523.
  11. Murakami D, Yamato M, Nishida K, Ohki T, Takagi R, Yang J, Namiki H, Okano T: Fabrication of transplantable human oral mucosal epithelial cell sheets using temperature-responsive culture inserts without feeder layer cells. J Artif Organs 2006;9:185–191.
  12. Yang J, Yamato M, Kohno C, Nishimoto A, Sekine H, Fukai F, Okano T: Cell sheet engineering: recreating tissues without biodegradable scaffolds. Biomaterials 2005;26:6415–6422.
  13. Yamada N, Okano T, Sakai H, Karikusa F, Sawasaki Y, Sakurai Y: Thermo-responsive polymeric surfaces; control of attachment and detachment of cultured cells. Makromol Chem Rapid Commun 1991;11:571–576.

    External Resources

  14. Ohki T, Yamato M, Murakami D, Takagi R, Yang J, Namiki H, Okano T, Takasaki K: Treatment of oesophageal ulcerations using endoscopic transplantation of tissue-engineered autologous oral mucosal epithelial cell sheets in a canine model. Gut 2006;55:1704–1710.
  15. Hirao M, Masuda K, Asanuma T, Naka H, Noda K, Matsuura K, Yamaguchi O, Ueda N: Endoscopic resection of early gastric cancer and other tumors with local injection of hypertonic saline-epinephrine. Gastrointest Endosc 1988;34:264–269.
  16. Gotoda T, Kondo H, Ono H, Saito Y, Yamaguchi H, Saito D, Yokota T: A new endoscopic mucosal resection procedure using an insulation-tipped electrosurgical knife for rectal flat lesions: report of two cases. Gastrointest Endosc 1999;50:560–563.
  17. Oyama T, Tomori A, Hotta K, Morita S, Kominato K, Tanaka M, Miyata Y: Endoscopic submucosal dissection of early esophageal cancer. Clin Gastroenterol Hepatol 2005;3:S67–70.
  18. Gotoda T, Yamamoto H, Soetikno RM: Endoscopic submucosal dissection of early gastric cancer. J Gastroenterol 2006;41:929–942.
  19. Katada C, Muto M, Manabe T, Boku N, Ohtsu A, Yoshida S: Esophageal stenosis after endoscopic mucosal resection of superficial esophageal lesions. Gastrointest Endosc 2003;57:165–169.
  20. Takagi R, Yamato M, Murakami D, Kondo M, Yang J, Ohki T, Nishida K, Kohno C, Okano T: Preparation of keratinocyte culture medium for the clinical applications of regenerative medicine. J Tissue Eng Regen Med 2011;5:e63–73.
  21. Takagi R, Murakami D, Kondo M, Ohki T, Sasaki R, Mizutani M, Yamato M, Nishida K, Namiki H, Yamamoto M, Okano T: Fabrication of human oral mucosal epithelial cell sheets for treatment of esophageal ulceration by endoscopic submucosal dissection. Gastrointest Endosc 2010;72:1253–1259.