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Clinical outcomes of xeno-free autologous cultivated
limbal epithelial transplantation: a 10-year study
Virender S Sangwan,
1,2
Sayan Basu,
1,2
Geeta K Vemuganti,
2
Kunjal Sejpal,
1
Sandhya V Subramaniam,
3
Souvik Bandyopadhyay,
4
Sannapaneni Krishnaiah,
5
Subhash Gaddipati,
2,6
Shubha Tiwari,
2,6
Dorairajan Balasubramanian
2,3,6
ABSTRACT
Purpose Ocular burns can damage the corneal epithelial
stem cells located at the limbus. This study evaluated
the efficacy of xeno-free autologous cell-based treatment
of limbal stem cell deficiency.
Methods This retrospective study included 200 patients,
above 8 years of age, with clinically diagnosed unilateral
total limbal stem cell deficiency due to ocular surface
burns treated between 2001 and 2010. A small limbal
biopsy was obtained from the unaffected eye. The limbal
epithelial cells were expanded ex vivo on human amniotic
membrane for 10e14 days using a xeno-free explant
culture system. The resulting cultured epithelial monolayer
and amniotic membrane substrate were transplanted on
to the patient’s affected eye. Postoperative corneal
surface stability, visual improvement and complications
were objectively analysed.
Results A completely epithelised, avascular and
clinically stable corneal surface was seen in 142 of 200
(71%) eyes at a mean follow-up of 361.6 (range:
1e7.6) years. A two-line improvement in visual acuity,
without further surgical intervention, was seen in 60.5%
of eyes. All donor eyes remained healthy.
Conclusions Autologous cultivated limbal epithelial
transplantation using a xeno-free explant culture
technique was effective in long-term restoration of
corneal epithelial stability and improvement of vision in
eyes with ocular surface burns.
INTRODUCTION
The corneal epithelium is continuously renewed by
a steady supply of cells from the palisades of Vogt,
located at the limbus.
1
There is now enough
scientific evidence to support that adult corneal
epithelial stem cells reside at this location.
23
Damage to the limbus by injury or disease, most
commonly due to ocular surface burns, leads to
limbal stem cell deficiency (LSCD) and conse-
quently corneal epithelial breakdown.
4
This clini-
cally manifests as recurrent or persistent epithelial
defects, progressive corneal vascularisation and
eventually loss of corneal clarity.
4
LSCD can be treated by transplanting healthy
limbal cells from the unaffected fellow eye (autol-
ogous) or from donors (allogeneic) depending on
whether the injury is unilateral or bilateral.
5
Over
the last two decades, the surgical technique has
evolved from direct limbal transplantation
67
to
transplantation of ex vivo cultivated limbal epithe-
lial cells,
8
which requires less donor tissue and hence
is thought to be safer for the donor eye. Despite this
advantage, limbal cultivation may involve use of
xenogenic tissue, associated with the risk of trans-
mission of known or unknown infections to the
transplant recipient.
9e11
Xenogenic constituents of
a limbal culture system may be in the form of murine
feeder cells,
81213
fetal bovine serum (FBS)
812e15
or
animal-derived growth factors.
16
Although an effective xeno-free technique of
limbal cultivation is desirable, only six groups have
reported the use of such a technique, but in few
eyes and with variable follow-up.
17e22
Currently,
there are no large studies on the long-term
outcomes of xeno-free autologous cultivated limbal
epithelial transplantation.
910
To fill this gap in
existing literature, we report the long-term
survival, visual benefit and complications of trans-
plantation of autologous limbal epithelial cells,
cultivated using a completely xeno-free explant
culture technique, in 200 eyes with LSCD following
ocular surface burns.
METHODS
Study design and subjects
The study was approved by the Institutional
Review Board of L V Prasad Eye Institute, Hyder-
abad, India, and was conducted in strict adherence
to the tenets of the Declaration of Helsinki. Prior
written informed consent was obtained from all
patients or guardians, as appropriate. A retrospec-
tive chart review of all patients who underwent
cultivated limbal epithelial transplantation for the
treatment of LSCD between 1 April 2001 and
1 April 2010 was carried out.
The inclusion criteria for this study were as
follows: (A) patients with a documented history of
chemical or thermal burns; (B) patients with age at
injury of more than 8 years; (C) patients who
underwent autologous limbal transplantation for
unilateral (defined as no clinical signs of ocular
surface disease in the other eye) and total LSCD
(defined as 3608superficial corneal vascularisation,
diffuse fluorescein staining of the corneal surface
with or without persistent epithelial defects,
conjunctivalisation of the corneal surface and
absence of limbal palisades of Vogt, figure 1); (D)
patients with a postoperative follow-up time $12
months. The following exclusion criteria were
applied: (A) patients who had bilateral LSCD or
had allogeneic limbal transplantation; (B) patients
who had limbal transplantation for partial LSCD or
LSCD due to causes other than ocular surface
burns; (C) cases treated prior to October 2002 when
FBS was used for limbal culture if autologous serum
(AS) was not obtained during limbal biopsy; (D)
1
Cornea and Anterior Segment
Services, L V Prasad Eye
Institute, Hyderabad, India
2
Sudhakar and Sreekanth Ravi
Stem Cell Biology Laboratory,
L V Prasad Eye Institute,
Hyderabad, India
3
Professor Brien Holden Eye
Research Center, L V Prasad Eye
Institute, Hyderabad, India
4
Indian Institute of Public
Health, Hyderabad, India
5
Department of Clinical
Epidemiology and Biostatistics,
L V Prasad Eye Institute,
Hyderabad, India
6
Champalimaud Translational
Center for Eye Research, L V
Prasad Eye Institute, Hyderabad,
India
Correspondence to
Dr Virender S Sangwan, L V
Prasad Eye Institute, Kallam Anji
Reddy Campus, Road No.2,
Banjara Hills, Hyderabad
500034, India;
vsangwan@lvpei.org
Accepted 9 July 2011
Published Online First
2 September 2011
Br J Ophthalmol 2011;95:1525e1530. doi:10.1136/bjophthalmol-2011-300352 1525
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patients with stable corneal epithelium, but with <1-year
follow-up; (E) patients with total LSCD, but with dry eye
disease (Schirmer’s test without anaesthesia of <10 mm in
5 min), were not considered for surgery; (F) patients with no
visual potential as determined by clinical examination and
electrophysiological testing (flash visual evoked potential and
flash electroretinogram); (G) patients with untreated concurrent
problems, such as glaucoma and infection, were not considered
for surgery.
Data collection
The data retrieved from the medical records included age and sex
of the patient, type and date of injury, details of prior ocular
procedures, Snellen’s best spectacle-corrected visual acuity
(BCVA) and intraocular pressure at presentation and at each
follow-up visit, presence or absence of lid abnormalities, dry eye
disease, symblepharon, degree of limbal involvement, intra-
operative surgical details, postoperative complications, duration
of follow-up and status of ocular surface at each visit (slit-lamp
findings including fluorescein staining).
Outcome measures
The primary outcome measure was success of transplantation,
defined clinically as a completely epithelised, avascular and clin-
ically stable corneal surface. Failure was defined as the occurrence
of superficial corneal vascularisation or persistent epithelial
defects. Survival time was calculated in months from the date of
limbal transplantation to the date of failure or the date of last
follow-up depending on the clinical outcome. The secondary
outcome measure was the proportion of eyes that had a two-line
improvement in BCVA at last follow-up or before undergoing
penetrating keratoplasty (PK) as compared to baseline.
Technique of limbal biopsy
A biopsy was taken from a healthy part of the limbus. A
232 mm piece of conjunctival epithelium with 1 mm into clear
corneal stromal tissue at the limbus was dissected. Conjunctiva
was excised just behind the pigmented line (palisades of Vogt),
and the limbal tissue that contained epithelial cells and a part of
the corneal stroma was obtained.
23
Technique of limbal culture
The tissue was transported to the laboratory in human
corneal epithelium (HCE) medium.
23
HCE is composed of
modified Eagle’s medium/F12 medium (1:1) solution containing
10% (vol/vol) AS, 2 mM l-glutamine, 100 U/ml penicillin,
100 mg/ml streptomycin, 2.5 mg/ml amphotericin B, 10 ng/ml
human recombinant epidermal growth factor and 5 mg/ml
human recombinant insulin.
23
Under strict aseptic conditions,
the donor limbal tissue was shredded into small pieces. Human
amniotic membrane (hAM), prepared and preserved by our eye
bank, was used as a carrier. A 334 cm hAM sheet was de-
epithelised using 0.25% recombinant trypsin
23
and EDTA solu-
tion for 15 min. The shredded bits of limbal tissue were
explanted over the centre of de-epithelised hAM with the
basement membrane side up. A similar parallel culture was also
prepared as a backup. A submerged explant culture system
without a feeder cell layer was used. We used the HCE medium
to nurture the culture. The culture was incubated at 378C with
5% CO
2
and 95% air. The growth was monitored daily under an
inverted phase contrast microscope, and the medium was
changed every other day. The culture was completed when
a monolayer of the cells growing from the explants became
confluent, typically in 10 to 14 days.
Figure 1 Clinical photographs of eyes before and after autologous cultivated limbal epithelial transplantation. Eyes of four different patients with total
limbal stem cell deficiency and variable amounts of corneal stromal scarring (A to D); same eyes 1 year after limbal transplantation (E to H). Right eye
of a 26-year-old female patient with a history of alkali injury showing a stable corneal surface, minimal stromal scarring and a best-corrected visual
acuity (BCVA) of 20/30 (E). Right eye of a 19-year-old male patient with a history of acid injury showing a stable corneal surface with residual stromal
scarring with a BCVA of 20/100 (F). Right eye of a 45-year-old male patient with a history of alkali injury showing recurrence of conjunctivalisation
inferiorly (failure) between 4 and 7 o’clock at the limbus, with a BCVA of 20/40 (G). Right eye of a 37-year-old female patient with a history of acid
injury showing a stable corneal surface and a clear corneal graft with a BCVA of 20/20 (H).
1526 Br J Ophthalmol 2011;95:1525e1530. doi:10.1136/bjophthalmol-2011-300352
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Technique of limbal transplantation
Any symblepharon which prevented adequate separation of
the lids was released to permit the insertion of a wire speculum
(no additional surgery to treat the symblepharon was
performed).
15
A peritomy was performed, and the corneal
fibrovascular pannus was excised. If excessive corneal thinning
or perforation was noted at this stage, a lamellar or pene-
trating keratoplasty was performed.
24
The hAM and mono-
layer of cultivated limbal epithelial cells were spread over the
cornea, epithelial side up. The graft was then secured to the
peripheral cornea by interrupted, circumferential 10-0 nylon
sutures and to the surrounding conjunctival edge by inter-
rupted 8-0 polyglactin sutures.
15
Alternately, using a sutureless
technique, the graft was secured to underlying ocular surface
with fibrin glue (TISSEEL Kit from Baxter AG, Austria), and
the margins of the graft were tucked under the surrounding
conjunctival edge.
15
Bandage contact lenses were not applied
at the end of surgery.
Postoperative management
All patients were treated with 1% prednisolone acetate eye drops
eight times a day tapered to once a day in 35e42 days and 0.3%
ciprofloxacin hydrochloride eye drops four times a day for
1 week. The latter were continued, as needed, if an epithelial
defect was present. No systemic antibiotics or steroids were
administered to any patients. Patients were examined on post-
operative days 1, 7 and 42 and at an interval of 90e180 days
thereafter, as customised by the clinical appearance of the
transplant. Each examination included a complete history, visual
acuity assessment with Snellen’s charts, intraocular pressure
measurement and detailed ocular examination with slit-lamp
biomicroscopy.
Statistical analysis
MedCalc (V.11.4.3.0) statistical software was used for data
analysis. The outcome of transplantation was analysed using
KaplaneMeier survival curves, and survival probability/rate at
1 year was reported as percentage (6SE). Multiple logistic
regression analysis was performed to test the association
between graft failure and clinical variables. A two-tailed p value
of <0.05 was considered statistically significant.
RESULTS
Demographics
This study included 200 eyes of 200 patients with LSCD due
to ocular burns. The mean age of the patients was
24.169.9 years, and the male to female ratio was 159:41.
Table 1 summarises the baseline demographic features of all
200 eyes. Although 56 (23.5%) fellow eyes had history of
simultaneous injury, none had clinical features of ocular
surface disease at presentation. The mean follow-up was 361.6
(range 1e7.6) years. All 200 patients had follow-up of 1 year;
117, 73, 61, 39, 18 and 4 patients had follow-up of 2, 3, 4, 5, 6
and 7 years, respectively.
Primary outcome
At final follow-up visit, 142 of the 200 (71%) eyes maintained
a completely epithelised, avascular and clinically stable corneal
surface. The KaplaneMeier survival rate was 76.4%63% at
1 year and 68.1%64% at 2 years and thereafter (figure 2).
Multiple logistic regression showed that eyes with prior amni-
otic membrane grafts (OR 3, 95% CI: 1.3 to 6.7, p¼0.007) or
prior keratoplasty (OR 4.7, 95% CI:1.9 to 11.3, p¼0.0006) or
keratoplasty along with limbal transplantation (OR 9.8, 95% CI:
1.9 to 50.1, p¼0.006) had greater risk of failure, but there was no
association seen with age, gender, duration between injury and
surgery, aetiology of injury, presence of lid abnormalities, prior
lid surgeries, presenting vision and presence or absence of
symblepharon (p>0.5).
Secondary outcome
Of the 200 eyes, a two-line improvement in BCVA was seen in
121 (60.5%) eyes. At last follow-up visit or before undergoing
keratoplasty, 80 (40%) eyes had BCVA of 20/60 or better, 54
(27%) had BCVA of 20/70 to 20/200 and 66 (33%) eyes had
BCVA worse than 20/200. Of the 66 eyes with BCVA of less
than 20/200, 58 (29%) eyes had poor vision due to recurrence of
LSCD, and 8 (4%) eyes had corneal scarring for which PK was
done subsequently.
Treatment failures and adverse events
Failures occurred between 1 month and 20.6 months of limbal
transplantation, and 47 (81%) of the 58 failures occurred within
1 year. Haemorrhage under the amniotic membrane was seen
postoperatively in 56 (23%) eyes, all of which resolved sponta-
neously. Among the 58 eyes with treatment failure, persistent
epithelial defects developed in 13 (22.4%) eyes; five (2.5%) eyes
developed sterile corneal melts which were managed with tissue
adhesive application. Three (1.5%) eyes developed bacterial
keratitis, of which two eyes resolved with intensive medical
Table 1 Demographic features of 200 eyes treated with
xeno-free autologous cultivated limbal epithelial
transplantation
Characteristic N (%)
Laterality
Right eye 111 (55.5)
Left eye 89 (44.5)
Aetiology of ocular burn
Alkali 107 (53.5)
Acid 55 (26.5)
Thermal 21 (11.5)
Unknown chemical 17 (8.5)
Ocular surgery prior to CLET
Amniotic membrane graft 87 (43.5)
Penetrating or lamellar keratoplasty 19 (9.5)
Symblepharon release 13 (6.5)
Entropion correction 11 (5.5)
Tarsorrhaphy 9 (4.5)
Tissue adhesive application 9 (4.5)
Cataract extraction 3 (1.5)
Best-corrected visual acuity before CLET
200/100 to 20/100 51 (25.5)
Worse than 20/200 149 (74.5)
Lid abnormalities
Upper lid entropion 7 (3.5)
Lower lid entropion 4 (2)
Trichiasis 5 (2.5)
Incomplete lid closure 9 (4.5)
Symblepharon present in at least one quadrant 100 (50)
Duration between injury and CLET
3 months to 6 months 11 (5.5)
6 months to 1 year 156 (78)
More than 1 year 33 (16.5)
Type of surgery performed
CLETs 100 (50)
CLET with symblepharon release 90 (45)
CLET with lamellar or penetrating keratoplasty 10 (5)
CLET, Cultivated limbal epithelial transplantation.
Br J Ophthalmol 2011;95:1525e1530. doi:10.1136/bjophthalmol-2011-300352 1527
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treatment and one eye needed an emergency PK. Successful ex
vivo expansion of the limbal epithelial cells was seen in all cases,
and none of the patients had to undergo a repeat biopsy. None of
the donor eyes developed any clinical signs or symptoms of
ocular surface disease, and the donor site healed without scarring
in 10e14 days (figure 3).
DISCUSSION
Previous studies have shown that cultivated limbal epithelial
transplantation is as effective as direct limbal transplantation for
the treatment of LSCD, while requiring less donor limbal tissue
and hence being safer for the donor eye.
6e10
However, limbal
cultivation is not without its disadvantages, like additional
laboratory costs and theoretical risks associated with using
xenobiotic materials for culture.
9e11
These two factors have
restricted the popularity of cultivated limbal epithelial trans-
plantation to a few advanced centres around the globe.
910
Having used FBS prior to October 2002,
15
we subsequently
developed a feeder-free explant culture system using human AS
and human recombinant growth factors.
23
Over the course of
the last decade, we have characterised the cultured cells,
25
standardised our cultivation technique
23
and shown how the
transplanted monolayer of cells forms a normal stratified corneal
phenotype in vivo.
24 26 27
Our previous report summarised the
clinical results in the initial 88 eyes with partial and total LSCD
due to a variety of causes, treated with limbal cells cultivated
using either FBS or AS, with a success rate of 73% and follow-up
ranging from 3 to 41 months.
15
In contrast to our previous
report, this large series of 200 eyes focused on a more homoge-
nous group with total LSCD due to ocular burns and
a minimum follow-up of 1 year.
This study found that the transplantation of limbal epithelial
cells cultivated using our xeno-free explant culture technique
was successful in ocular surface restoration in 71% of the
200 recipient eyes. Previous studies by Nakamura et al,
17
Shimakazi et al,
18
Di Girolamo et al,
19
Kolli et al
20
and Zakaria et
al
22
also described xeno-free techniques for autologous limbal
cultivation, but they were limited by the relatively smaller
samples of two to eight eyes. In addition to being clinically
effective and free of animal-derived products, this technique of
cultivation was also extremely reliable, as ex vivo expansion was
successful in every case. We have previously shown that
Figure 2 KaplaneMeier survival analysis of 200 eyes treated with
autologous cultivated limbal epithelial transplantation from April 2001 to
April 2010. The survival was 76.5%63% at 1 year postoperatively and
68.1%64% at 2 years and thereafter.
Figure 3 Serial clinical photographs of donor eyes of two different patients after limbal biopsy showing different morphological patterns of
epithelisation. Right eye of a 23-year-old male patient shows a large epithelial defect involving the bulbar conjunctiva and limbus 1 day after limbal
biopsy. (A) Three days later, the limbus had re-epithelised, and the conjunctival defect had reduced. (B) A small v-shaped defect on the bulbar
conjunctiva was noted 7 days after biopsy, (C) and complete epithelisation was seen 2 weeks after biopsy (D). Left eye of a 19-year-old female patient
shows a similar large epithelial defect involving the bulbar conjunctiva and limbus 1 day after limbal biopsy (A); within 3 days, the limbus had re-
epithelised with reduction in the size of the conjunctival defect (B). A small oval defect remained on the bulbar conjunctiva 7 days after biopsy (C), and
complete epithelisation was noted 14 days after biopsy, on the day of limbal transplantation in the contralateral eye (D).
1528 Br J Ophthalmol 2011;95:1525e1530. doi:10.1136/bjophthalmol-2011-300352
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inadequate growth or contamination is seen in <1.5% of the
cultures in our system.
28
The overall success rate of cultivated limbal epithelial trans-
plantation, as reported in various studies, ranges between 50%
and 100%.
9 10
In comprehensive reviews of this procedure, Baylis
et al and Shortt et al found no significant differences in the
clinical outcomes based on source of donor tissue (autologous or
allogeneic), culture technique (explant or suspension) or indi-
cation for surgery.
910
Caution must be exercised before
comparing our results with those of existing literature because
the indications for surgery, sample size and follow-up duration
are widely variable among different studies.
89
It is noteworthy
in this context that with similar indications for surgery, clinical
criteria for success and follow-up, our success rate (71%, 200
eyes) compares well with that of Rama et al (68%, 107 eyes)
12
and Di Iorio et al (80%, 166 eyes),
13
who unlike us used a feeder-
cell- and FBS-based suspension culture technique.
The actual mechanism by which limbal transplantation
works is still debated. It is unclear whether this treatment
replenishes the stem cell reserve
12
or revives the surviving stem
cells by improving the microenvironment.
10
It is also widely
accepted that the cause of failure of limbal transplantation is
multifactorial and poorly understood.
910
Surprisingly, we found
that eyes having undergone prior amniotic membrane grafts or
PK were more prone to failure of the limbal transplant. This
finding needs to be interpreted carefully and should not be
misconstrued as a contraindication for performing these proce-
dures during acute stage of ocular burns. This association is
simply indicative of the fact that eyes with more severe injury
need multiple surgical interventions and are more difficult to
treat.
12
Additionally, we found that the timing of limbal trans-
plantation did not affect the eventual outcome. In our opinion,
this procedure can be performed as early as 3 months after the
initial injury, but only if the ocular surface inflammation has
been adequately controlled by that time.
The strengths of this study include the large, homogenous
sample and long follow-up. The limitations include the
retrospective design and subjective clinical criteria for diag-
nosis and success of treatment. The rarity of the disease
makes a retrospective design a necessity, although a multi-
centric prospective study involving all the clinical centres
around the world practicing this treatment is something
worth considering. The alternative to clinical assessment is an
objective scoring system based on impression cytology or
a symptom-based questionnaire, both of which have inherent
limitations.
In conclusion, our goal was to study the long-term clinical
outcomes of transplanting autologous limbal epithelial cells,
cultivated using a xeno-free explant culture system for eyes with
LSCD due to ocular burns. We found that our technique was
successful in long-term restoration of the ocular surface and
vision with no adverse effect on the donor eyes. This report
provides ample clinical evidence to support the use of simpler
xeno-free limbal cultures, which will eliminate any real or
perceived risks associated with the use of animal products and
allow wider applicability of this effective technique.
Funding This work was funded by a competitive grant from the Department of
Biotechnology, India (BT/01/COE/06/02/10), a partnership grant from the
Champalimaud Foundation, Portugal, and support from Sudhakar and Sreekanth Ravi,
California, USA (for equipment and laboratory infrastructure). None of these had any
role in the design, collection, analysis and interpretation of the data.
Competing interests None.
Patient consent Obtained.
Ethics approval Ethics Committee, L V Prasad Eye Institute, Hyderabad, India.
Contributors All authors included on the paper fulfil the criteria of authorship. In
addition, there is no one else who fulfils the criteria but has not been included as an
author. On behalf of all coauthors, Virender S Sangwan, MS, states that he had full
access to all the data in the study and takes responsibility for the integrity of the data
and the accuracy of the data analysis.
Provenance and peer review Not commissioned; externally peer reviewed.
REFERENCES
1. Shapiro MS, Friend J, Thoft RA. Corneal re-epithelialization from the conjunctiva.
Invest Ophthalmol Vis Sci 1981;21:135e42.
2. Schermer A, Galvin S, Sun TT. 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;103:49e62.
3. Shanmuganathan VA, Foster T, Kulkarni BB, et al. Morphological characteristics of
the limbal epithelial crypt. Br J Ophthalmol 2007;91:514e19.
4. Dua HS, Azuara-Blanco A. Limbal stem cells of the corneal epithelium. Surv
Ophthalmol 2000;44:415e25.
5. Fernandes M, Sangwan VS, Rao SK, et al. Limbal stem cell transplantation. Indian J
Ophthalmol 2004;52:5e22.
6. Kenyon KR, Tseng SC. Limbal autograft transplantation for ocular surface disorders.
Ophthalmology 1989;96:709e22.
7. Tan DT, Ficker LA, Buckley RJ. Limbal transplantation. Ophthalmology
1996;103:29e36.
8. Pellegrini G, Traverso CE, Franzi AT, et al. Long-term restoration of damaged
corneal surfaces with autologous cultivated corneal epithelium. Lancet
1997;349:990e3.
9. Baylis O, Figueiredo F, Henein C, et al. 13 years of cultured limbal epithelial cell
therapy: a review of the outcomes. J Cell Biochem 2011;112:993e1002.
10. Shortt AJ, Secker GA, Notara MD, et al. Transplantation of ex vivo cultured limbal
epithelial stem cells: a review of techniques and clinical results. Surv Ophthalmol
2007;52:483e502.
11. Schwab IR, Johnson NT, Harkin DG. Inherent risks associated with manufacture
of bioengineered ocular surface tissue. Arch Ophthalmol 2006;124:1734e40.
12. Rama P, Matuska S, Paganoni G, et al. Limbal stem-cell therapy and long-term
corneal regeneration. N Engl J Med 2010;363:147e55.
13. Di Iorio E, Ferrari S, Fasolo A, et al. Techniques for culture and assessment of limbal
stem cell grafts. Ocul Surf 2010;8:146e53.
14. Baradaran-Rafii A, Ebrahimi M, Kanavi MR, et al. Midterm outcomes of autologous
cultivated limbal stem cell transplantation with or without penetrating keratoplasty.
Cornea 2010;29:502e9.
15. Sangwan VS, Matalia HP, Vemuganti GK, et al. Clinical outcome of autologous
cultivated limbal epithelium transplantation. Indian J Ophthalmol 2006;54:29e34.
16. Pauklin M, Fuchsluger TA, Westekemper H, et al. Midterm results of cultivated
autologous and allogeneic limbal epithelial transplantation in limbal stem cell
deficiency. Dev Ophthalmol 2010;45:57e70.
17. Nakamura T, Inatomi T, Sotozono C, et al. Transplantation of autologous serum-
derived cultivated corneal epithelial equivalents for the treatment of severe ocular
surface disease. Ophthalmology 2006;113:1765e772.
18. Shimazaki J, Higa K, Morito F, et al. Factors influencing outcomes in cultivated
limbal epithelial transplantation for chronic cicatricial ocular surface disorders. Am J
Ophthalmol 2007;143:945e53.
19. Di Girolamo N, Bosch M, Zamora K, et al. A contact lens-based technique for
expansion and transplantation of autologous epithelial progenitors for ocular surface
reconstruction. Transplantation 2009;87:1571e8.
20. Kolli S, Ahmad S, Lako M, et al. Successful clinical implementation of corneal
epithelial stem cell therapy for treatment of unilateral limbal stem cell deficiency.
Stem Cells 2010;28:597e610.
21. Meller D, Fuchsluger T, Pauklin M, et al. Ocular surface reconstruction in graft-
versus-host disease with HLA-identical living-related allogeneic cultivated limbal
epithelium after hematopoietic stem cell transplantation from the same donor.
Cornea 2009;28:233e6.
22. Zakaria N, Koppen C, Van Tendeloo V, et al. Standardized limbal epithelial stem
cell graft generation and transplantation. Tissue Eng Part C Methods
2010;16:921e7.
23. Mariappan I, Maddileti S, Savy S, et al. In vitro culture and expansion of human
limbal epithelial cells. Nat Protoc 2010;5:1470e9.
24. Basu S, Mohamed A, Chaurasia S, et al. Clinical outcomes of penetrating
keratoplasty after autologous cultivated limbal epithelial transplantation for ocular
surface burns. Am J Ophthalmol 2011. Accepted for publication 23 May 2011.
25. Polisetti N, Agarwal P, Khan I, et al. Gene expression profiles of epithelial cells and
mesenchymal cells derived from limbal explant culture. Mol Vis 2010;16:1227e40.
26. Sangwan VS, Matalia HP, Vemuganti GK, et al. Early results of penetrating
keratoplasty after cultivated limbal epithelium transplantation. Arch Ophthalmol
2005;123:334e40.
27. Fatima A, Vemuganti GK, Iftekhar G, et al. In vivo survival and stratification of
cultured limbal epithelium. Clin Experiment Ophthalmol 2007;35:96e8.
28. Fatima A, Sangwan VS, Iftekhar G, et al. Technique of cultivating limbal derived
corneal epithelium on human amniotic membrane for clinical transplantation.
J Postgrad Med 2006;52:257e61.
Clinical science
Br J Ophthalmol 2011;95:1525e1530. doi:10.1136/bjophthalmol-2011-300352 1529
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2011 95: 1525-1529 originally published onlineBr J Ophthalmol
Virender S Sangwan, Sayan Basu, Geeta K Vemuganti, et al.
10-year study acultivated limbal epithelial transplantation:
Clinical outcomes of xeno-free autologous
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