Skip to main content
Springer Nature Link
Log in

cG-CAOMECS—clinical-grade cultured autologous oral mucosal epithelial cell sheet

  • Regular Article
  • Published:
Cell and Tissue Research Aims and scope Submit manuscript

Abstract

The present study reports the feasibility and successful production of rabbit cG-CAOMECS, designed to reconstruct corneal epithelium of patients with bilateral limbal stem cell deficiency. To produce a safe, chemically defined and FDA compliant cG-CAOMECS, oral mucosal epithelial cells were isolated from a biopsy of rabbit buccal tissue and seeded on a cGMP-certified cell culture surface coated with GMP-grade extracellular matrix. A newly designed clinical-grade medium (KaFa™ medium) was utilized to carry out cell expansion. Detachment and harvesting of the produced cell sheet was accomplished using collagenase treatment. Live cell imaging and morphological analysis techniques were used to examine cell growth. Cells attached onto the surface and self-assembled into colony-forming units (CFUs). Microscopic examination showed that CFUs formed during the first 5 days, and basal monolayer cell sheet formed in less than 10 days. Cells expanded to form a multilayered epithelial cell sheet that was harvested after 17–19 days in culture. Immunostaining and Western blot analyses showed that deltaNp63 was expressed in the basal cells and K3/K12 was expressed in the apical cells, indicating the presence of corneal epithelial-like cells in the produced cell sheet. Adhesion molecules, E-cadherin, beta-catenin, and Cnx43 were also expressed and exhibited the epithelial integrity of the cell sheet. The expression of integrin-beta1 and beta4 confirmed that the collagenase treatment used for detaching and harvesting the cell sheet did not have adverse effects. Our results showed that the utilization of clinical-grade and FDA-approved reagents successfully supported the production of cG-CAMECS.

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

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Explore related subjects

Discover the latest articles and news from researchers in related subjects, suggested using machine learning.

References

  • Ballen PH (1963) Mucous membrane grafts in chemical (lye) burns. Am J Ophthalmol 55:302–312. https://doi.org/10.1016/0002-9394(63)92687-4

  • Burillon C, Huot L, Justin V, Nataf S, Chapuis F, Decullier E, Damour O (2012) Cultured autologous oral mucosal epithelial cell sheet (CACAOMECS) transplantation for the treatment of corneal limbal epithelial stem cell deficiency. Invest Ophthalmol Vis Sci 53(3):1325–1331. https://doi.org/10.1167/iovs.11-7744

  • Denig R (1912) A surgical treatment for alkali burns of the eye. Munchen Med Wochenschr 1:579–590

    Google Scholar 

  • Bardag-Gorce F, Makalinao A, Meepe I, Hoft RH, Cortez D, Oliva J, Niihara Y (2018) Corneal keratin aggresome (CKAGG) formation and clearance by proteasome activation. Heliyon 4(12):e01012. https://doi.org/10.1016/j.heliyon.2018.e01012.

  • Bardag-Gorce F, Oliva J, Wood A, Hoft R, Pan D, Thropay J, Makalinao A, French SW, Niihara Y (2015) Carrier-free cultured autologous oral mucosa epithelial cell sheet (CAOMECS) for corneal epithelium reconstruction: a histological study. Ocul Surf Apr 13(2):150–163. https://doi.org/10.1016/j.jtos.2014.12.003

    Article  Google Scholar 

  • Bardag-Gorce F, Oliva J, Wood A, Niihara H, Makalinao A, Sabino S, Niihara Y (2013) Microarray analysis of oral mucosal epithelial cell sheet. Tissue Eng Regen Med 10:362-370

  • Bardag-Gorce F, Hoft RH, Wood A, Oliva J, Niihara H, Makalinao, A, Niihara Y (2016) The role of E-cadherin in maintaining the barrier function of corneal epithelium after treatment with cultured autologous oral mucosa epithelial cell sheet grafts for limbal stem deficiency. J Ophthalmol 4805986. https://doi.org/10.1155/2016/4805986

  • Gallico GG 3rd, O’Connor NE, Compton CC, Kehinde O, Green H (1984) Permanent coverage of large burn wounds with autologous cultured human epithelium. N Engl J Med 311:448–451

    Article  Google Scholar 

  • Gallico GG 3rd, O’Connor NE, Compton CC, Remensnyder JP, Kehinde O, Green H (1989) Cultured epithelial auto grafts for giant congenital nevi. Plast Reconstr Surg 84:1–9

    Article  Google Scholar 

  • Gipson IK, Geggel HS, Spurr-Michaud SJ (1986) Transplant of oral mucosal epithelium to rabbit ocular surface wounds in vivo. Arch Ophthalmol Oct 104(10):1529–1533 (PMID: 3767686)

  • Hassan NT, AbdelAziz NA (2018) Oral mucosal stem cells, human immature dental pulp stem cells and hair follicle bulge stem cells as adult stem cells able to correct limbal stem cell deficiency. Curr Stem Cell Res Ther 13(5):356–361. https://doi.org/10.2174/1574888X13666180223124936

    Article  CAS  PubMed  Google Scholar 

  • Hayashida Y, Nishida K, Yamato M, Watanabe K, Maeda N, Watanabe H, Kikuchi A, Okano T, Tano Y (2005) Ocular surface reconstruction using autologous rabbit oral mucosal epithelial sheets fabricated ex vivo on a temperature-responsive culture surface.. Invest Ophthalmol Vis Sci 46(5):1632–1639. PMID: 15851562

  • Higa K, Shimazaki J (2008) Recent advances in cultivated epithelial transplantation. Cornea Sep 27(Suppl 1):S41–S47

    Article  Google Scholar 

  • Hirayama M, Satake Y, Higa K, Yamaguchi T, Shimazaki J (2012) Transplantation of cultivated oral mucosal epithelium prepared in fibrin-coated culture dishes. Invest Ophthalmol Vis Sci 53(3):1602–1609

  • Kinoshita S, Koizumi N, Nakamura T (2004) Transplantable cultivated mucosal epithelial sheet for ocular surface reconstruction. Exp Eye Res Mar 78(3):483–491

    Article  CAS  Google Scholar 

  • Jones KB, Klein OD (2013) Oral epithelial stem cells in tissue maintenance and disease: the first steps in a long journey. Int J Oral Sci Sep 5(3):121–129. https://doi.org/10.1038/ijos.2013.46

    Article  CAS  Google Scholar 

  • Lee J, Shin D, Roh JL (2018) Use of a pre-vascularised oral mucosal cell sheet for promoting cutaneous burn wound healing. Theranostics Eight 20:5703–5712. https://doi.org/10.7150/thno.28754

    Article  CAS  Google Scholar 

  • Ma DH, Kuo MT, Tsai YJ, Chen HC, Chen XL, Wang SF, Li L, Hsiao CH, Lin KK (2009) Transplantation of cultivated oral mucosal epithelial cells for severe corneal burn. Eye (Lond) 23(6):1442–1450. https://doi.org/10.1038/eye.2009.60. PMID: 19373264

  • Melotti A, Mas C, Kuciak M, Lorente-Trigos A, Borges I, Altaba RI, A, (2014) The river blindness drug Ivermectin and related macrocyclic lactones inhibit WNT-TCF pathway responses in human cancer. EMBO Mol Med Oct Six 10:1263–1278. https://doi.org/10.15252/emmm.201404084

    Article  CAS  Google Scholar 

  • Nakamura T, Endo K, Cooper LJ, Fullwood NJ, Tanifuji N, Tsuzuki M, Koizumi N, Inatomi T, Sano Y, Kinoshita S (2003) The successful culture and autologous transplantation of rabbit oral mucosal epithelial cells on amniotic membrane. Invest Ophthalmol vis Sci Jan 44(1):106–116

    Article  Google Scholar 

  • Nakamura T, Kinoshita S (2003) Ocular surface reconstruction using cultivated mucosal epithelial stem cells. Cornea 22(7 Suppl):S75–S80

  • Nishida K, Yamato M, Hayashida Y, Watanabe K, Yamamoto K, Adachi E, Nagai S, Kikuchi A, Maeda N, Watanabe H, Okano T, Tano Y (2004) Corneal reconstruction with tissue engineered cell sheets composed of autologous oral mucosal epithelium. N Engl J Med 351(12):1187–1196

    Article  CAS  Google Scholar 

  • Priya CG, Arpitha P, Vaishali S, Prajna NV, Usha K, Sheetal K, Muthukkaruppan V (2011) Adult human buccal epithelial stem cells: identification, ex-vivo expansion, and transplantation for corneal surface reconstruction. Eye (lond) 25(12):1641–1649

    Article  CAS  Google Scholar 

  • Rama P, Matuska S, Paganoni G, Spinelli A, De Luca M, Pellegrini G (2010) Limbal stem-cell therapy and long-term corneal regeneration. N Engl J Med 363(2):147–155

    Article  CAS  Google Scholar 

  • Boneva RS, Folk TM, Chapman LE (2001) Infectious disease issues in xenotransplantation. Clin Microbiol Rev 14(1):1–14. https://doi.org/10.1128/CMR.14.1.1-14.2001

  • Rovere MR, Rousselle P, Haftek M, Charleux B, Kocaba V, Auxenfans, C, Nataf S, Damour O (2018) Preserving basement membranes during detachment of cultivated oral mucosal epithelial cell sheets for the treatment of total bilateral limbal stem cell deficiency Cell Transplant 27(2):264-274 https://doi.org/10.1177/0963689717741140

  • Sorkio A, Hongisto H, Kaarniranta K, Uusitalo H, Juuti-Uusitalo K, Skottman H (2014) Structure and barrier properties of human embryonic stem cell–derived retinal pigment epithelial cells are affected by extracellular matrix protein coating. Tissue Eng Part A 20(3–4):622–634. https://doi.org/10.1089/ten.tea.2013.0049

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Utheim TP, Utheim ØA, Khan Q-E-S, Sehic A (2016) Culture of oral mucosal epithelial cells for the purpose of treating limbal stem cell deficiency. J Funct Biomater 1;7(1)

  • Yamaguchi N, Isomoto H, Kobayashi S, Kanai N, Kanetaka K, Sakai Y, Kasai Y, Takagi R, Ohki T, Fukuda H, Kanda T, Nagai K, Asahina I, Nakao K, Yamato M, Okano T, Eguchi S (2017). Oral epithelial cell sheets engraftment for esophageal strictures after endoscopic submucosal dissection of squamous cell carcinoma and airplane transportation. Sci Rep 7(1):17460. https://doi.org/10.1038/s41598-017-17663-w

Download references

Author information

Authors and Affiliations

  1. The Lundquist Institute At Harbor UCLA Medical Center, Torrance, CA, 90502, USA

    Kavita Narwani, Jeremy Stark, Daileen Cortez, Isaac Yang, Christian Au, Alissa Diaz, Catalina Guerra, Yutaka Niihara & Fawzia Bardag-Gorce

  2. Emmaus Medical, 21250 Hawthorne Blvd, Suite 800, Torrance, CA, 90503 , USA

    Yutaka Niihara

Authors
  1. Kavita Narwani
    View author publications

    Search author on:PubMed Google Scholar

  2. Jeremy Stark
    View author publications

    Search author on:PubMed Google Scholar

  3. Daileen Cortez
    View author publications

    Search author on:PubMed Google Scholar

  4. Isaac Yang
    View author publications

    Search author on:PubMed Google Scholar

  5. Christian Au
    View author publications

    Search author on:PubMed Google Scholar

  6. Alissa Diaz
    View author publications

    Search author on:PubMed Google Scholar

  7. Catalina Guerra
    View author publications

    Search author on:PubMed Google Scholar

  8. Yutaka Niihara
    View author publications

    Search author on:PubMed Google Scholar

  9. Fawzia Bardag-Gorce
    View author publications

    Search author on:PubMed Google Scholar

Corresponding author

Correspondence to Fawzia Bardag-Gorce.

Ethics declarations

Ethics approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC).

Informed consent

This article does not contain any studies with human participants performed by any of the authors.

Conflict of interest

Grant support: Research is fully supported by Emmaus Medical Inc. Dr. Fawzia Bardag Gorce and Mrs. Kavita Narwani salaries were fully supported by Emmaus Medical Inc., and Dr. Yutaka Niihara is the CEO and the Chairman of Emmaus Medical, Inc. The rest of the authors have no support from Emmaus Medical, Inc. and no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Narwani, K., Stark, J., Cortez, D. et al. cG-CAOMECS—clinical-grade cultured autologous oral mucosal epithelial cell sheet. Cell Tissue Res 386, 47–57 (2021). https://doi.org/10.1007/s00441-021-03507-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00441-021-03507-7

Keywords