José Reinaldo da Silva Ricardo,MD Telma Pereira Barreiro,MD PHD Priscila Cristovam Cardoso, BHS Rossen Myhailov Hazarbassanov, MD Myrna Serapião dos Santos, MD,PHD José Alvaro Pereira Gomes, MD,PHD The authors have no financial interest in the subject matter of this poster.

Purpose To report the clinical results of transplantation of conjunctival epithelial cells cultivated ex vivo in patients with total limbal stem cell deficiency (TLSCD). Methods Design: Prospective, noncomparative, interventional case series. Participants: Twelve eyes of 10 patients with clinical and cytological diagnosis of TLSCD. Ethical approval was obtained from the Institutional Medical Ethics Committee and National Ethics Committee in Research, and followed the Tenets of the Declaration of Helsinki. Informed consent was obtained from all patients before treatment.

________________________ Methods Main Outcome Measures were graded 1,2 preoperatively and again at 6 months postoperatively: Clinical parameters:  Corneal opacity:1. total transparency, 2. haze, 3. partial view of the iris, and 4. no view of the iris.  Epithelial integrity : 1. no fluorescein staining, 2. superficial punctate keratitis, 3. erosion or small epithelial defect, 4. extensive epithelial defect.  Corneal superficial neovascularization : 1. peripheral neovascularization, 2. peripheral and midperipheral neovascularization, 3. modest neovascularization involving the entire cornea, 4. massive neovascularization of the entire cornea.  Visual acuity: best-corrected visual acuity in the affected eye was measured at each clinical visit.  Impression cytology and cytokeratin profiles (CK3 /CK19)  In vivo corneal confocal microscopy

________________________________________________________ Methods Autologous conjunctival epithelial cells were cultured in amniotic membrane submerged in culture medium (DMEM/ F12 ).The cultivated tissue was transplanted to the recipient eye after superficial keratectomy. (Figure 1) 1D1D 1A1A 2A2A 1A1B1C 1D 1E 1F1G1H Figure 1. Conjunctival epithelial cells transplantation cultured ex vivo in a patient with total limbal deficiency. 1A) Patient with total limbal deficiency (pre-operative). 1B) Conjunctival biopsy performed in superior forniceal conjunctiva. 1C) Explant was placed on the culture medium in a denuded amniotic membrane. 1D) Expansion and cell confluence after 15 days.1E) Superficial keratectomy. 1F) The ex vivo cultured epithelial sheets are cut from the culture insert using a blade 15, and transferred over the prepared corneal surface. 1G) Mechanical protection is achieved using an amniotic membrane. 1H) Final aspect at 6 months postoperatively showed a regular and integrity corneal epithelium.

________________________ Methods  The changes in clinical parameters of TLSCD such as improvement in visual acuity, corneal phenotype as determined by impression cytology and cytokeratin profiling, and findings on corneal confocal microscopy were correlated to determine whether results for outcomes were consistent.  Complete success was defined as an improvement in the clinical parameters of LESC deficiency and the restoration of a more normal corneal epithelial phenotype on impression cytology in all quadrants of the cornea.  Partial success was defined as failure of the limbus in 1 or 2 quadrants of the cornea based on clinical and impression cytology analysis.  Total failure was defined as no improvement in clinical parameters and impression cytology findings in more than three quadrants of the cornea.

CaseAge(yrs)ConditionPrevious procedures Pre-VACOSNEIICConfocal 145 Chemical burnIrCLALHM 333 ConjunctivalN/P 243 Multiple surgeriesPTG(4x)CF 333 N/P 320 Chemical burnSimb+OM+GSHM 444 N/P 437 SJSKLAL(2x)HM 444 N/P 525 PAS-HM 333 ConjuctivalN/P 667 Idiophatic-HM 444 Conjunctival 734 SJSSimb+OM+GSLP 444 Conjunctival 847 Chemical burnLRCLAL+PK+AMTHM 444 Conjunctival 964 Multiple surgeriesPKLP 444 Conjunctival 1047 Chemical burn-HM 444 Conjunctival 1167 Idiophatic-HM 444 Conjunctival 1277 Idiophatic-LP 333 Conjunctival Results The mean follow up time was 12.1 months (range, 6-20 months). Clinical outcomes for all patients are presented in Table 1. Table 1. Pre-operative characteristics of patients treated. AMT = amniotic membrane transplant ; CO= corneal opacity; CF= counter finger; EI= epithelial integrity; F= female; IC=impression cytology; HM= hand motion; KLAL = keratolimbal allograft ;LRCLAL= living related conjunctival limbal allograft; LP= light perception; M= male; N/P= not possible; OM= oral mucosa; PAS= polyglandular autoimmune syndrome; PK = penetrating keratoplasty; PTG= excision of ptergium; SN= superficial vascularization; SG= salivary gland; Simb= simblepharoplasty; SJS = Stevens–Johnson syndrome; yrs= years; VA= visual acuity.

____________________________________________________ Results Table 3. Clinical Outcomes After Surgical Transplantation of Ex Vivo Cultured Conjunctival Epithelial Sheets in 12 Eyes With Total Limbal Stem Cell Deficiency COEISN meanSDmeanSDmeanSD Preoperative Postoperative 2.42 ● ● ● 0.57 CO=corneal opacity; EI=epithelial Integrity; SN=superficial neovascularization; SD=standard deviation; Wilcoxon: ● P<0.01. Figure 2. Slit-lamp photographs of four patients take before surgery (A-D-G-J) and at 6 months follow-up without fluorescein (B-E-H-L) and with fluorescein (C- F-I-M). The cornea surface was covered with clear corneal epithelium. Patient 2 (A-C), patient 3 (D-F), patient 5 (G-I) and patient 6 (J-M). The regenerated corneal surface appeared transparent and regular, with characteristics of corneal epithelium. (Figure 2) Parameters of stem cell failure were graded preoperatively and again at 6 months postoperatively. (Table 3)

___________________________________________ Results Visual acuity improved in 7 of 12 eyes (58.3 %) to the range of hand movements to 0.5. (Figure 3) NLP LP HM CF NL 0.01 CF HM LP NLP Preoperative Visual Acuity Final Visual Acuity Figure 3. Changes in Visual Acuity after Surgery. NLP denotes no light perception, HM hand motion, and CF counting fingers. The diagonal line indicates the values at which the preoperative and postoperative values for visual acuity were the same.

_______________________________________________________________ Results  In postoperative evaluation, 3/8 eyes (37. 5%) showed the corneal phenotype and 5/8 (62. 5%) displayed a mixture of both conjunctival and corneal phenotypes. (Figure 4)  CK3 expression was positive in 38.27% preoperatively and 50.97% postoperatively.  CK19 expression in 46.58% preoperatively and in 41.61% postoperatively.  In vivo confocal analysis confirmed the clinical and cytological findings. A B E F CD G H D Figure 4. Confocal microscopy and impression cytology with HE/PAS and immunocytochemistry to CK3 and 19 preoperatively (A-D) and 6 months post-operatively (E-H). BCD H

The overall success rate for this treatment in our cohort was 10/12 (83.3%), where complete success was achieved in 8 patients (66.7%) and partial success in 2 patients (16.7%) in a mean follow-up time of 12.1 months (range, 6-20 months). Table 4. Postoperative outcomes CaseFollow-up (m) Pos-VACOSNEIICConfocal microscopy ComplicationsClinical outcomes 120CF221N/PCorneal-Success 22020/40211N/PCorneal-Success 318HM311N/P -Success 417HM433CornealN/PyesFailure 51120/125211N/P -Success 61120/200121Corneal -Success 79CF324N/P yesFailure 89HM321MixedCorneal-Partial 99HM322MixedCorneal-Success 10920/200221MixedCorneal-Success 116HM222MixedCorneal-Success 126HM222MixedCorneal-Partial CF= counter finger; EI= epithelial integrity; CO=corneal opacity; M= months; N/P= not possible; SN= superficial neovascularization; VA= visual acuity;

Conclusions  The mean follow-up of 12 months (range 6-20 months) allowed us to validate the clinical findings after the first 6 months of the surgery. The regenerated corneal surface appeared transparent and regular, with characteristics of corneal epithelium. Impression cytology, cytokeratin profile and confocal microscopy helped us to correlate the clinical findings with objective measures in most of the cases.  We showed that transplanted ex vivo cultivated conjunctival epithelial cells (EVCAU) produced clinical results with a success rate of 83.3% equivalent to that of transplanted cultivated corneal epithelium reported in the literature (mean of 75.4%, range of 33%-100%). 3  In bilateral cases, the transplantation of EVCAU can be considered as the first option with the advantages of using an autologous source of tissue: it has a reduced risk of graft rejection and transmission of infection, and reduces the need for long-term steroids or immunosuppression. Moreover, only a small amount of autologous conjunctival tissue is required. Even in cases of bilateral involvement, as long as a small amount of healthy conjunctiva is available (1 mm x 2 mm), we are still able to cultivate ex vivo an epithelial sheet of sufficient size to resurface the corneal defect. 2

Conclusions  We demonstrated the effectiveness of transplantation of conjunctival epithelial cells cultivated ex vivo for corneal surface reconstruction in cases with TLSCD. Future studies are needed to further assess the long-term efficacy of this procedure. References  1.Inatomi T, Nakamura T, Kojyo M, et al. Ocular surface reconstruction with combination of cultivated autologous oral mucosal epithelial transplantation and penetrating keratoplasty. Am J Ophthalmol 2006;142(5):  2.Ang LP, Tanioka H, Kawasaki S, et al. Cultivated human conjunctival epithelial transplantation for total limbal stem cell deficiency. Invest Ophthalmol Vis Sci 2010;51(2):  3. 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;52:483–502.