Current Eye Research

ISSN: 0271-3683 (Print) 1460-2202 (Online) Journal homepage: http://www.tandfonline.com/loi/icey20

Xenobiotic- and Serum-Free Culture of Oral

Mucosal Epithelial Cells on Contact Lenses
Benny Bjrkblom, Jon R. Eidet, Tor P. Utheim, Harald F. Ulltveit-Moe & Sten
Raeder
To cite this article: Benny Bjrkblom, Jon R. Eidet, Tor P. Utheim, Harald F. Ulltveit-Moe & Sten
Raeder (2016) Xenobiotic- and Serum-Free Culture of Oral Mucosal Epithelial Cells on Contact
Lenses, Current Eye Research, 41:1, 20-27, DOI: 10.3109/02713683.2015.1004720
To link to this article: http://dx.doi.org/10.3109/02713683.2015.1004720

Published online: 06 Feb 2015.

Submit your article to this journal

Article views: 153

View related articles

View Crossmark data

Full Terms & Conditions of access and use can be found at

http://www.tandfonline.com/action/journalInformation?journalCode=icey20
Download by: [36.79.24.163]

Date: 24 January 2016, At: 13:24

Current Eye Research, 2016; 41(1): 2027

Copyright ! Taylor & Francis Group, LLC
ISSN: 0271-3683 print / 1460-2202 online
DOI: 10.3109/02713683.2015.1004720

ORIGINAL ARTICLE

Xenobiotic- and Serum-Free Culture of Oral Mucosal

Epithelial Cells on Contact Lenses
Benny Bjorkblom1*, Jon R. Eidet2*, Tor P. Utheim2,3, Harald F. Ulltveit-Moe4 and
Sten Raeder4,5
Department of Chemistry, Umea University, Umea, Sweden, 2Department of Medical Biochemistry,
Oslo University Hospital, Oslo, Norway, 3Department of Oral Biology, Faculty of Dentistry, University of Oslo,
Oslo, Norway, 4Department of Ophthalmology, Stavanger University Hospital, Stavanger, Norway and
5
The Norwegian Dry Eye Clinic, Oslo, Norway

Downloaded by [36.79.24.163] at 13:24 24 January 2016

ABSTRACT
Purpose/aim: Cultured autologous oral mucosal epithelial cells (OMECs) have proven useful in the treatment
of ocular surface disorders. This study is the first to investigate the potential of expanding OMEC in a
xenobiotic- and serum-free medium using therapeutic contact lenses (CLs) as a substrate and carrier.
Materials and methods: Porcine OMEC were seeded on laminin-coated lotrafilcon A therapeutic CLs with
the density of 8  104 cells/lens and cultured in a defined serum and xenobiotic-free medium. Confocal
immunofluorescence microscopy was used to analyze the following: (1) cellular morphology by using
rhodamine-phalloidin staining of F-actin, (2) phenotype by applying antibodies against the progenitor cell
marker p63 and the putative stem cell marker ABCG2 and (3) cell viability by using propidium iodide and
Hoechst 33342 dual staining.
Results: Porcine OMEC attached well to the CLs, and cell-to-cell contacts were evident. After three days
in culture, the OMEC displayed a confluent monolayer with uniform cobblestone morphology, whereas
stratified cultures with 23 layers were formed after six days. No significant difference in expression of p63 was
observed after three-day culture (79.4 14.8%) compared with six-day culture (60.3 18.9%). ABCG2 expression
in the basal cell layer was 6.3 1.0% and 4.8 1.8% after three- and six-day culture, respectively. The basal layer
viability of cultured OMECs was 99.3 0.2% and 82.8 1.1% after three and six days culture, respectively.
Conclusions: The use of therapeutic CLs has potential as a substrate and carrier for OMEC cultured in a
xenobiotic- and serum-free culture system.
Keywords: Cell culture, oral mucosa, contact lens, epithelial cells

INTRODUCTION

most severe forms result in blindness. In addition,

LSCD may be a painful. LSCD can be treated by
transplantation of cultured human limbal epithelial
cells.3 However, in bilateral severe LSCD, other
sources than limbal cells must be used if immunesuppressive medications are to be avoided. There is
no consensus on the duration of such medications and
prolonged, and even lifelong, immunosuppression
has been suggested.4 Therefore, the search for alternative cell sources to treat LSCD is of paramount

The surface of the cornea, the epithelium, is renewed

by stem cells located at the peripheral limbal region.1,2
These cells can be harmed by a number of factors,
including chemical burns, infections, autoimmune
diseases and a number of genetic diseases, which
may result in limbal stem cell deficiency (LSCD).
LSCD is characterized by ingrowth of conjunctival
epithelial cells and vessels onto the cornea, and the

*These authors contributed equally to this work.

Received 18 June 2014; revised 10 December 2014; accepted 22 December 2014; published online 5 February 2015
Correspondence: Jon Roger Eidet, MD, PhD, Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway. E-mail:
j.r.eidet@gmail.com

20

Downloaded by [36.79.24.163] at 13:24 24 January 2016

Culture of Oral Mucosa on Contact Lenses

importance. So far, there have only been clinical
studies on the use of conjunctival5 and oral mucosal
epithelial cells (OMECs) to treat LSCD.6 In animal
models, however, a number of other cell types have
been used. Of all alternative non-limbal cell types
available, only oral mucosal cells have been broadly
documented to have clinical effect.624 As most causes
of LSCD do not affect oral mucosal cells, these cells
may be used for culture and subsequent transplantation. Even in StevensJohnson syndrome, an
autoimmune disease that affects both the eyes and
the oral mucosa, transplantation of cultured OMEC
has proved successful.17 In addition to avoiding
immune reactions, the use of autologous OMEC has
the benefit of avoiding any risk of transferring
infectious agents.
Over the past 15 years, many cell substrates have
been proposed for ocular surface reconstruction.
Amniotic membrane has been the most used substrate.25 Although it has several benefits, including
being anti-immunogenic, anti-scarring and growthstimulating, its drawbacks include labor intensive
preparation, suboptimal optical transmission, risk of
disease transmission and batch-to-batch variability.25
All human- or animal-derived carriers, including
collagen and fibrin, may present a possibility of
infection. Due to these disadvantages, it is of great
interest to develop synthetic scaffolds for transplanting cells onto eyes with LSCD. The usefulness of
contact lenses (CLs) for transfer of cultured cells in
LSCD has already been demonstrated.5,2628 However,
to our knowledge, the culture of OMECs on CLs has
never previously been reported. Moreover, this study
is the first to use a completely serum- and xenobioticfree culture medium without applying amniotic
membrane. Collectively, these measures make our
study the first to apply a completely defined cultured
protocol. In that, we comply with the increasingly
stricter regulatory demands, which aim at reducing
(and preferably completely avoid) the risk of transferring known (e.g. bovine spongiform encephalitis) and
hitherto unknown diseases.29 By reducing variation
from culture to culture to an absolute minimum, our
protocol enables standardization of the treatment of
LSCD. Apart from regulatory issues, standardization
of culture protocols is important to make comparisons
between treatment modalities for LSCD easier, which
is considered an important goal to advance corneal
regenerative medicine.25

MATERIALS AND METHODS

Preparation of Porcine OMEC Cultures
Pigs (Fatland Jaeren AS, Rogaland, Norway) used in
this study were treated in accordance with the ARVO
Statement for the Use of Animals in Ophthalmic and
Copyright ! 2016 Taylor & Francis Group, LLC

21

Vision Research. This study was approved by the

Regional Committee for Medical and Health Research
Ethics, Norway. Porcine oral mucosal tissue was
obtained within one hour post mortem. A total of 12
trephinations from the buccal oral cavity from each
animal were made using 6-mm biopsy punches (Kai
Industries, Gifu, Japan). The harvested tissue was
washed in Dulbeccos Minimal Essential Medium,
50 IE penicillin, 50 mg/ml streptomycin and 2.5 mg/l
amphotericin B (Sigma-Aldrich, St Louis, MO). The
submucosal connective tissue was removed with
scissors to the extent possible; the samples were
then incubated with 1.2 IU dispase II (Roche
Diagnostics, Basel, Switzerland) to separate the epithelium from the remaining connective tissue.
Thereafter, a single cell suspension was obtained by
exposing the epithelium to 0.25% trypsin-EDTA
(Sigma-Aldrich) followed by filtrating the suspension
through a 40 mm pore size cell strainer (BD
Biosciences, San Jose, CA).

Culture of Porcine OMECs on Therapeutic

CLs
Lotrafilcon A soft therapeutic CL (AirOptix
Night&Day, CIBA VISION, Duluth, GA) were coated
with laminin (Millipore Corporation, Billerica, MA)
by immersing the CL in phosphate buffered saline
(Sigma-Aldrich) containing 24 mg/ml laminin for one
hour at 37  C. Laminin is an extracellular matrix
glycoprotein and is the main non-collagenous component of basal lamina that supports adhesion, proliferation and differentiation of many cell types
including epithelial cells. The CL were stabilized
with the concavity facing up in 24-well culture plates
during culture by using custom-made polydimethylsiloxane CL-holders (Figure 1). OMECs were seeded
8  104 cells/well as determined by automated cell
counting
(ScepterTM,
Millipore)
in
CnT-24
(CELLnTEC, Bern, Switzerland), and the culture
medium was replaced after two and four days of
culture. CnT-24 is a liquid culture medium kit
including both basal medium and separate supplements. It is fully defined and has low calcium
(0.07 mM). It does not contain bovine pituitary extract
(BPE), serum or antibiotics/antimycotics. The basal
medium contains amino acids, minerals, vitamins and
organic compounds, but is protein free.

Immunofluorescence
OMECs grown on CLs were fixed in 4% (w/v)
buffered paraformaldehyde for 30 min at room temperature (RT) followed by permeabilization in 0.5%
Triton X-100 for one minute. Unspecific binding of
antibody to specimen was blocked by incubation in

22 B. Bjorkblom et al.

Downloaded by [36.79.24.163] at 13:24 24 January 2016

FIGURE 1 (A) Technical drawing of the custom made contact lens (CL) holders used to enable cell culturing on therapeutic CLs.
(B) Lotrafilcon A CLs placed with the concavity facing up in the CL holders that were customized to fit inside a standard well of a
24-well plate with 16 mm well diameter and 3.5 ml total well volume. Approximately, 80,000 OMEC were seeded onto the concavity
of each CL and cultured in 1 ml CnT-24 serum-free culture medium. (C) Phase-contrast image of seeded OMEC as described in (B).

10% fetal bovine serum (Invitrogen, Carlsbad, CA),

0.2% Tween-20 (Sigma-Aldrich) for one hour prior to
primary antibody incubation, 10 mg/ml mouse antip63 (clone 4A4, Sigma-Aldrich) or 12.5 mg/ml mouse
anti-ABCG2 (B7059, Sigma-Aldrich) for one hour at
RT. The samples were incubated with 4 mg/ml antimouse Alexa 488 (Invitrogen) secondary antibody for
one hour at RT prior to labeling with 5 U/ml
rhodamine phalloidin (Invitrogen) for 30 min at
RT and 2 mg/ml Hoechst 33342 (Invitrogen) DNA
stain. The cells were finally mounted onto glass
slides in Mowiol mounting media containing 2.5%
(w/v) Dabco (Sigma-Aldrich) anti-fading reagent.
Fluorescence images were captured using an
inverted Nikon A1R confocal laser scanning microscope (Tokyo, Japan) with a 60 oil immersion
objective lens. Excitation of the fluorescent probes
Hoechst 33342, Alexa 488 and rhodamine phalloidin,
and propidium iodide (PI) were done using laser
lines 408 nm, 488 nm or 561 nm, respectively and
images recorded at 425/475 nm, 500/550 nm or
570/620 nm, respectively. For viability measurements, the cells were stained in growth media
containing 10 mg/ml PI (Sigma-Aldrich) for 30 min
at 37  C prior to fixation in paraformaldehyde and
Hoechst 33342 staining as above. Quantification of
cell viability by Hoechst 33342/PI double labeling
was done using a Zeiss Axioplan 2 fluorescent
microscope equipped with ISIS Imaging System V
5.2 imaging software (MetaSystems, Altlussheim,
Germany). For both phenotype and viability assessment, the percentage of positive cells was calculated
based on the average of positive cells of a total of 100
cells judged by two independent investigators
blinded to the experimental design.

Statistical Analyses
Students t-test was used to compare the groups.
Data were expressed as mean standard error of the

mean. A p
significant.

value

of 50.05

was

considered

RESULTS
To determine the usefulness of CnT-24 as a culture
medium combined with therapeutic CLs as an ex vivo
cell growth support and transport system for OMEC,
we investigated the morphology, viability and phenotype of cultured OMEC after three and six days,
respectively.

Morphology of OMECs Cultured on

Therapeutic CLs
The isolated epithelial cells attached well to the
therapeutic CLs and exhibited a confluent cobblestone
monolayer after three days in culture (Figure 2A).
Limited stratification (23 cell layers) was observed
following six days in culture (Figure 2B). Irrespective
of the culture time, the cells were uniform, and cellto-cell contacts were evident (Figure 2C). Rarely, a few
loosely attached cells were observed superficially.

Phenotype of OMECs Cultured on

Therapeutic CLs
The phenotype of the cultured cells was analyzed
by immunostaining with p63 and ABCG2. p63 is a
transcription factor that is commonly used to identify
progenitor cells,30 whereas ABCG2 is a ATP-binding
cassette transporter protein that is suggested to be a
potential stem cell marker31 (Figure 3A and B). p63
expression following three and six days of culture was
79.4 14.8% and 60.3 18.9%, respectively (p = 0.079;
n = 6 in each group). Expression of ABCG2 after three
and six days of culture was 6.3% and 4.8%, respectively (p = 0.119; n = 6 in each group; Figure 3C).
Current Eye Research

Downloaded by [36.79.24.163] at 13:24 24 January 2016

Culture of Oral Mucosa on Contact Lenses

23

FIGURE 2 (A) Fluorescent microscopy images of OMECs stained with rhodamine phalloidin to visualize the microfilaments and cell
shape after three and six days of culture. The dotted lines indicate the boarder of the contact lenses (CLs). Scale bar = 100 mm. (B)
Confocal laser scanning microscopy images of OMEC stained for microfilament (rhodamine phalloidin) and nuclei (Hoechst 33342) at
three and six days of culture. Scale bar = 10 mm. (C) A single x, y confocal microscopy scan of OMEC grown on CLs at day 6. Combined
z stacks from the indicated x, y area (crossed lines) showing cell layer stratification. Cells stained as in (B). Scale bar = 20 mm.

Viability of OMECs Cultured on Therapeutic

CLs
Viability of the cultured cells was assessed by PI and
Hoechst 33342 DNA double labeling (Figure 4A).
Cells with nuclei positively labeled with PI were
scored as necrotic/late apoptotic. Cell viability was
99.3 0.2% after three days and dropped to 82.8 1.1%
after six days (p50.001; n = 6 in each group;
Figure 4B).

DISCUSSION
In this study, we demonstrated a xenobiotic- and
serum-free culture system using cultured OMEC on
therapeutic CLs. Confocal immunofluorescence
microscopy after three-day culture displayed a confluent cobblestone morphology, expression of the
progenitor cell marker p63 and the putative stem
cell marker ABCG2, and high viability.
Copyright ! 2016 Taylor & Francis Group, LLC

A monolayer and 23 layers of cells were observed

after three and six days of culture in our study,
respectively. This is in contrast to a study by
Gaddipati et al.11 demonstrating a monolayer even
after nine days of culture of OMEC. Differences in the
culture protocols, especially the use of cell suspension
compared to explant culture in the Indian study, may
explain the contradictory results. It is well known that
at least two days of culture are required before visible
outgrowth from explants can be observed.11 It is likely
that increased culture time in our study would
increase the stratification as seen in several studies
on cultured OMEC using cell suspension with7,13 or
without air-lifting.14
If a non-CL approach had been chosen, an epithelium of at least 23 layers of cells would generally be
regarded more advantages than a monolayer as a
confluent and well-stratified epithelium is expected to
have increased resistance to infections.32 Another
argument often stated for the necessity of a wellstratified epithelium is the fact that the transplanted

Downloaded by [36.79.24.163] at 13:24 24 January 2016

24 B. Bjorkblom et al.

FIGURE 3 Representative confocal laser scanning microscopy images of OMEC cultured for three and six days after staining with
filamentous actin microfilaments and Hoechst 33342. (A) Immunostaining with antibody against p63. Scale bar = 20 mm. (B)
Immunostaining with antibody against ABCG2. Scale bar = 20 mm. (C) Percentage expression of cells stained with p63 and ABCG2
following three and six days of culture. Mean values SEM from six independent donors are shown.

cells should have sufficient mechanical strength to

endure the surgery and the postoperative period.33,34
However, as the cells in our study are cultured onto a
CL, we assume that the cells will better endure the
surgery and postoperative period compared to traditional culture methods on amniotic membrane where
the cells are more exposed. As the cultured cells also
need to be rearranged on the denuded corneal surface
following the transfer from the CLs, there is yet no
consensus on the optimal number of cell layers when
CLs are used.
Interestingly, Deshpande et al. observed the formation of a multilayer epithelium about 11 d after
CL-based transfer of a monolayer of cells in vitro 3D
rabbit organ culture model. This study indicates that
transplantation of one cell layer may be sufficient.

The most striking example of in vivo culture, however,

is Simple Limbal Epithelial Transplantation where
several non-cultured minute explants are placed onto
an amniotic membrane sutured to the cornea after
prior removal of the pannus. The technique has
proven highly successful in restoring the ocular
surface due to LSCD in several studies,3537 but
long-term results are awaiting. Furthermore, these
studies point to our three-day protocol as adequate for
clinical implementation, although it is significantly
shorter than traditional methods, requiring 23 weeks
of culture. Based on our high p63 content and
excellent viability at three days of culture, we suggest
this time interval as the optimal prior to transfer of the
cells. Clinical studies, however, are warranted to
evaluate the usefulness of our proposed method.
Current Eye Research

Culture of Oral Mucosa on Contact Lenses

25

Downloaded by [36.79.24.163] at 13:24 24 January 2016

FIGURE 4 (A) Representative confocal laser scanning microscopy images of OMEC at three and six days of culture showing viability
by staining with propidium iodide and Hoechst 33342. Scale bar = 20 mm. (B) Percentage expression of viable cells at three and six days
of culture. Mean values SEM from six independent donors are shown.

To analyze the phenotype of the cultured cells, the

number of cultured OMEC that expressed p63 and
ABCG2 was calculated. Although p63 expression has
been observed by immunostaining in several studies
on cultured OMEC,11,14,38,39 to our knowledge, only
the study by Oie et al. provide quantitative data.38 The
authors compared the effects of two types of
irradiated feeder layers (NIH/3T3 and dermal fibroblasts) on cultured OMEC.38 The percentage of p63positive cells after 1417 d of culture in the dermal
fibroblast group and NIH/3T3 group was 46% and
31%, respectively. The longer culture period compared to our study may explain the difference in
expression. This assumption is supported by: (1) a
study by Joseph et al., which revealed a decrease in
the number of p63-positive cells in the limbal explants
with increased culture time40 and (2) a paper by Kolli
et al., which showed lower p63 expression in limbal
epithelial cells with a peripheral location to the
explant compared to the central cells.41 In the peripheral zone of the sheet, the number of cell doublings is
higher than those centrally; however, the change in
p63 expression may also be due to the difference
in the distance to the limbal niche that may secrete
factors that prevent differentiation.41 The issue of
differentiation has been highlighted by a recent study
by Rama et al. demonstrating that increased differentiation leads to significantly poorer clinical results.42
It is promising that our culture system maintains a
high expression of p63 at both three and six days
of culture despite no use of amniotic membrane
as culture substrate, which has been proposed to serve
as a stem cell niche.43,44
To our knowledge, quantitative ABCG2 immunostaining of cultured OMEC has not been performed
previously, which makes direct comparisons impossible.9,15 However, RT-PCR data on ABCG2 of
cultured OMEC has recently been presented by
Copyright ! 2016 Taylor & Francis Group, LLC

Kolli et al., demonstrating a relatively low expression

of ABCG2 compared to limbal tissue.41 Thus, their
study is in line with our data showing low ABCG2
expression at both time intervals.
Over the past few years, there has been a trend
towards shorter culture periods for limbal stem cell
therapy.4,2527,39 The concept of culture in vivo
rather than in vitro partly26 or fully35 has recently
gathered worldwide attention as it significantly
eases corneal regenerative medicine. Moreover,
Hayashi et al. observed significantly lower viability
and reduced number of cell layers by increasing
the culture time (from 15 to 28 d). Unfortunately,
quantitative data on p63 expression was not
provided in their study.
Our choice of culture intervals was primarily based
on a study by Deshpande et al. demonstrating that
transfer of cultured cells onto CLs was improved by
shortening the culture time.26 In their study, human
epithelial cells cultured on coated CLs for up to five
days showed poor transfer unless cells were deliberately enzymatically removed using dispase. The
authors found one day of culture to be ideal to
secure transfer of cells onto the denuded surface of a
3D rabbit organ culture model. However, a recent
study by Brown et al. demonstrates successful transfer
in up to 81% of the cases even after four days of
culture.27
In conclusion, our study has demonstrated that
therapeutic CLs may be used as carriers for OMEC
expanded in a xenobiotic- and serum-free culture
system. Our study provides a promising platform for
a simplified and, most importantly, safer approach for
expansion of OMEC. As this is a cell culture study
only, future studies showing successful transplantation of OMEC cultured on CLs are needed to
demonstrate clinical applicability of the current protocol in the treatment of LSCD.

26 B. Bjorkblom et al.

ACKNOWLEDGMENTS
We would like to thank Rune Dysjaland at Fatland AS,
Oddmund Nordgaard and Emiel Jansson at
Laboratory for Molecular Biology, Stavanger
University Hospital, and Roger Eidet, for their excellent assistance and support.

DECLARATION OF INTEREST

Downloaded by [36.79.24.163] at 13:24 24 January 2016

The authors report no conflicts of interest. The authors

alone are responsible for the content and writing of
the paper.
This study was supported, in part, by Stavanger
University Hospital and the Eastern Norway Regional
Health Authority.

REFERENCES
1. Cotsarelis G, Cheng SZ, Dong G, Sun TT, Lavker RM.
Existence of slow-cycling limbal epithelial basal cells that
can be preferentially stimulated to proliferate: implications
on epithelial stem cells. Cell 1989;57:201209.
2. Davanger M, Evensen A. Role of the pericorneal papillary
structure in renewal of corneal epithelium. Nature 1971;
229:560561.
3. Pellegrini G, Traverso CE, Franzi AT, Zingirian M,
Cancedda R, De Luca M. Long-term restoration of
damaged corneal surfaces with autologous cultivated
corneal epithelium. Lancet 1997;349:990993.
4. Utheim TP. Limbal epithelial cell therapy: past, present,
and future. Methods Mol Biol 2013;1014:343.
5. Di Girolamo N, Bosch M, Zamora K, Coroneo MT,
Wakefield D, Watson SL. A contact lens-based technique
for expansion and transplantation of autologous epithelial
progenitors
for
ocular
surface
reconstruction.
Transplantation 2009;87:15711578.
6. Nishida K, Yamato M, Hayashida Y, Watanabe K,
Yamamoto K, Adachi E, et al. Corneal reconstruction
with tissue-engineered cell sheets composed of autologous
oral mucosal epithelium. N Engl J Med 2004;351:11871196.
7. Ang LP, Nakamura T, Inatomi T, Sotozono C, Koizumi N,
Yokoi N, Kinoshita S. Autologous serum-derived cultivated oral epithelial transplants for severe ocular surface
disease. Arch Ophthalmol 2006;124:15431551.
8. Burillon C, Huot L, Justin V, Nataf S, Chapuis F, Decullier
E, Damour O. Cultured autologous oral mucosal epithelial
cell sheet (CAOMECS) transplantation for the treatment of
corneal limbal epithelial stem cell deficiency. Invest
Ophthalmol Vis Sci 2012;53:13251331.
9. Chen HC, Chen HL, Lai JY, Chen CC, Tsai YJ, Kuo MT,
et al. Persistence of transplanted oral mucosal epithelial
cells in human cornea. Invest Ophthalmol Vis Sci 2009;50:
46604668.
10. Chen HC, Yeh LK, Tsai YJ, Lai CH, Chen CC, Lai JY, et al.
Expression of angiogenesis-related factors in human corneas after cultivated oral mucosal epithelial transplantation. Invest Ophthalmol Vis Sci 2012;53:56155623.
11. Gaddipati S, Muralidhar R, Sangwan VS, Mariappan I,
Vemuganti GK, Balasubramanian D. Oral epithelial cells
transplanted on to corneal surface tend to adapt to the
ocular phenotype. Indian J Ophthalmol 2013;62:644648.

12. Inatomi T, Nakamura T, Koizumi N, Sotozono C, Yokoi N,

Kinoshita S. Midterm results on ocular surface reconstruction using cultivated autologous oral mucosal epithelial
transplantation. Am J Ophthalmol 2006;141:267275.
13. Inatomi T, Nakamura T, Kojyo M, Koizumi N, Sotozono C,
Kinoshita S. Ocular surface reconstruction with combination of cultivated autologous oral mucosal epithelial
transplantation and penetrating keratoplasty. Am J
Ophthalmol 2006;142:757764.
14. Kolli S, Ahmad S, Mudhar HS, Meeny A, Lako M,
Figueiredo FC. Successful application of ex vivo expanded
human autologous oral mucosal epithelium for the treatment of total bilateral limbal stem cell deficiency. Stem
Cells 2014;32:21352146.
15. Ma DH, Kuo MT, Tsai YJ, Chen HC, Chen XL, Wang SF, et al.
Transplantation of cultivated oral mucosal epithelial cells
for severe corneal burn. Eye (Lond) 2009;23:14421450.
16. Nakamura T, Inatomi T, Cooper LJ, Rigby H, Fullwood NJ,
Kinoshita S. Phenotypic investigation of human eyes with
transplanted autologous cultivated oral mucosal epithelial
sheets for severe ocular surface diseases. Ophthalmology
2007;114:10801088.
17. Nakamura T, Inatomi T, Sotozono C, Amemiya T,
Kanamura N, Kinoshita S. Transplantation of cultivated
autologous oral mucosal epithelial cells in patients with
severe ocular surface disorders. Br J Ophthalmol 2004;88:
12801284.
18. Nakamura T, Kinoshita S. Ocular surface reconstruction
using cultivated mucosal epithelial stem cells. Cornea
2003;22:S75S80.
19. Nakamura T, Takeda K, Inatomi T, Sotozono C, Kinoshita
S. Long-term results of autologous cultivated oral mucosal
epithelial transplantation in the scar phase of severe ocular
surface disorders. Br J Ophthalmol 2011;95:942946.
20. Priya CG, Arpitha P, Vaishali S, Prajna NV, Usha K, Sheetal
K, Muthukkaruppan V. Adult human buccal epithelial
stem cells: identification, ex-vivo expansion, and transplantation for corneal surface reconstruction. Eye (Lond)
2011;25:16411649.
21. Satake Y, Dogru M, Yamane GY, Kinoshita S, Tsubota K,
Shimazaki J. Barrier function and cytologic features of the
ocular surface epithelium after autologous cultivated oral
mucosal epithelial transplantation. Arch Ophthalmol 2008;
126:2328.
22. Satake Y, Higa K, Tsubota K, Shimazaki J. Long-term
outcome of cultivated oral mucosal epithelial sheet transplantation in treatment of total limbal stem cell deficiency.
Ophthalmology 2011;118:15241530.
23. Sotozono C, Inatomi T, Nakamura T, Koizumi N, Yokoi N,
Ueta M, et al. Visual improvement after cultivated oral
mucosal epithelial transplantation. Ophthalmology 2013;
120:193200.
24. Takeda K, Nakamura T, Inatomi T, Sotozono C,
Watanabe A, Kinoshita S. Ocular surface reconstruction
using the combination of autologous cultivated oral
mucosal epithelial transplantation and eyelid surgery for
severe ocular surface disease. Am J Ophthalmol 2011;152:
195201 e191.
25. Utheim TP, Lyberg T, Raeder S. The culture of limbal
epithelial cells. Methods Mol Biol 2013;1014:103129.
26. Deshpande P, Notara M, Bullett NA, Daniels JT, Haddow
DB, Mac NS. Development of a surface modified contact
lens for transfer of cultured limbal epithelial cells for ocular
surface diseases. Tissue Eng Part A 2009;15:28892902.
27. Brown KD, Low S, Mariappan I, Abberton KM, Short R,
Zhang H, et al. Plasma polymer-coated contact lenses for
the culture and transfer of corneal epithelial cells in the
treatment of limbal stem cell deficiency. Tissue Eng Part A
2014;20:646655.
Current Eye Research

Downloaded by [36.79.24.163] at 13:24 24 January 2016

Culture of Oral Mucosa on Contact Lenses

28. Gore A, Horwitz V, Gutman H, Tveria L, Cohen L, CohenJacob O, et al. Cultivation and characterization of limbal
epithelial stem cells on contact lenses with a feeder layer:
toward the treatment of limbal stem cell deficiency. Cornea
2014;33:6571.
29. Schwab IR, Johnson NT, Harkin DG. Inherent risks
associated with manufacture of bioengineered ocular
surface tissue. Arch Ophthalmol 2006;124:17341740.
30. Pellegrini G, Dellambra E, Golisano O, Martinelli E,
Fantozzi I, Bondanza S, et al. p63 identifies keratinocyte
stem cells. Proc Natl Acad Sci U S A 2001;98:31563161.
31. De Paiva CC, Chen Z, Corrales RM, Pflugfelder SC,
Li DQ. ABCG2 transporter identifies a population of
clonogenic human limbal epithelial cells. Stem Cells 2005;
23:6373.
32. Klotz SA, Au YK, Misra RP. A partial-thickness epithelial
defect increases the adherence of Pseudomonas aeruginosa
to the cornea. Invest Ophthalmol Vis Sci 1989;30:10691074.
33. Koizumi N, Cooper LJ, Fullwood NJ, Nakamura T, Inoki K,
Tsuzuki M, Kinoshita S. An evaluation of cultivated
corneal limbal epithelial cells, using cell-suspension culture. Invest Ophthalmol Vis Sci 2002;43:21142121.
34. Koizumi N, Rigby H, Fullwood NJ, Kawasaki S, Tanioka H,
Koizumi K, et al. Comparison of intact and denuded
amniotic membrane as a substrate for cell-suspension
culture of human limbal epithelial cells. Graefes Arch Clin
Exp Ophthalmol 2007;245:123134.
35. Sangwan VS, Basu S, MacNeil S, Balasubramanian D.
Simple limbal epithelial transplantation (SLET): a novel
surgical technique for the treatment of unilateral limbal
stem cell deficiency. Br J Ophthalmol 2012;96:931934.
36. Bhalekar S, Basu S, Lal I, Sangwan VS. Successful autologous simple limbal epithelial transplantation (SLET) in

Copyright ! 2016 Taylor & Francis Group, LLC

37.

38.

39.

40.

41.

42.

43.

44.

27

previously failed paediatric limbal transplantation for

ocular surface burns. BMJ Case Rep 2013;10 May 2013.
Lal I, Panchal BU, Basu S, Sangwan VS. In-vivo expansion
of autologous limbal stem cell using simple limbal epithelial transplantation for treatment of limbal stem cell
deficiency. BMJ Case Rep 2013;22 May 2013.
Oie Y, Hayashi R, Takagi R, Yamato M, Takayanagi H, Tano
Y, Nishida K. A novel method of culturing human oral
mucosal epithelial cell sheet using post-mitotic human
dermal fibroblast feeder cells and modified keratinocyte
culture medium for ocular surface reconstruction. Br J
Ophthalmol 2010;94:12441250.
Hayashi R, Yamato M, Takayanagi H, Oie Y, Kubota A,
Hori Y, et al. Validation system of tissue-engineered
epithelial cell sheets for corneal regenerative medicine.
Tissue Eng Part C Methods 2010;16:553560.
Joseph A, Powell-Richards AO, Shanmuganathan VA,
Dua HS. Epithelial cell characteristics of cultured human
limbal explants. Br J Ophthalmol 2004;88:393398.
Kolli S, Lako M, Figueiredo F, Mudhar H, Ahmad S. Loss
of corneal epithelial stem cell properties in outgrowths
from human limbal explants cultured on intact amniotic
membrane. Regen Med 2008;3:329342.
Rama P, Matuska S, Paganoni G, Spinelli A, De Luca M,
Pellegrini G. Limbal stem-cell therapy and long-term
corneal regeneration. N Engl J Med 2010;363:147155.
Koizumi N, Inatomi T, Quantock AJ, Fullwood NJ, Dota A,
Kinoshita S. Amniotic membrane as a substrate for cultivating limbal corneal epithelial cells for autologous transplantation in rabbits. Cornea 2000;19:6571.
Grueterich M, Tseng SC. Human limbal progenitor cells
expanded on intact amniotic membrane ex vivo. Arch
Ophthalmol 2002;120:783790.