Allogeneic Cultivated Limbal Epithelial Sheet Transplantation in Reconstruction of Conjunctival Sac After Chemical and Thermal Burns.

The study aims to evaluate the effect of allogeneic cultivated limbal epithelial cell sheet transplantation (CLET) in reconstructing conjunctival sac for severe symblepharon after chemical and thermal burns. A retrospective, non-comparative case series. Thirty-six eyes (36 patients) underwent CLET for severe symblepharon and conjunctival sac stenosis or atresia. Symblepharon was separated, and pseudopterygium was preserved to replace the palpebral conjunctiva. Allogeneic cultivated limbal epithelial cell sheet using human amniotic membrane as a carrier was transplanted into the recipient's eye to reconstruct the conjunctival sac. The effect of conjunctival sac reconstruction, eye and eyelid movement, ocular surface restitution, and symblepharon recurrence were analyzed after surgery. Symblepharon was completely relieved in 30 of the 36 eyes (83.3%) by a single surgical procedure, with fornix reconstruction, as well as free movement of eye globe and eyelids. Strip-like symblepharon remained in 6 eyes (16.7%) and was completely relieved after the second CLET. Twenty patients without visual function received prostheses 3 months after surgery and the other sixteen patients underwent different corneal transplantation for visual acuity improvement. During the follow-up period, no one had symblepharon recurrence. The transplantation of cultivated allogeneic limbal epithelial sheets offers an effective and safe alternative in the treatment of symblepharon and reconstruction of conjunctival sac in eyes with severe ocular burns, which lays the foundation for subsequent treatments.

Transplantation of human corneal limbal epithelial cell sheet harvested on synthesized carboxymethyl cellulose and dopamine in a limbal stem cell deficiency.

This study aimed to evaluate the efficacy and safety of transplantation with human corneal limbal epithelial (HCLE) cell sheets cultured on carboxymethyl cellulose (CMC)-dopamine (DA)-coated substrates and harvested via enzymatic digestion of CMC with cellulase in a rabbit animal model of limbal stem cell deficiency (LSCD). Synthesized CMC-DA was pretreated onto the surface of culture plates. Then, HCLE cells were cultured on precoated CMC-DA and HCLE cell sheets were harvested using cellulase-containing cell culture medium. HCLE cell sheets were evaluated using a live/dead assay, histological examination, and immunofluorescence staining. For in vivo assessment, HCLE cell sheets were transplanted in a rabbit model of LSCD for 2 weeks to determine the effectiveness of the repair. Primary culture of HCLE cells stained positively for p63, cytokeratin (CK)15, and CK12. HCLE cell sheets were generated with a well-preserved morphology and transparency ranging in size from 15 to 19 mm after cellulase-assisted cell sheet generation. HCLE cell sheets uniformly stained positively for human mitochondria, p63, CK15, CK12, CK3/2p, and zonula occludens (ZO)-1. HCLE cell sheet transplantation in a rabbit model of LSCD improved the corneal opacity and neovascularization scores. Transplanted HCLE cell sheets stained positively for p63 and CK12. Transplantation of HCLE cell sheets harvested on CMC-DA coating combined with cellulase is a safe and efficient procedure for corneal epithelial regeneration in a rabbit model of LSCD. This system could enable a promising strategy to regenerate corneal epithelium by transplantation in ocular surface disorders.

Multi-parametric evaluation of autologous cultivated Limbal epithelial cell transplantation outcomes of Limbal stem cell deficiency due to chemical burn.

BACKGROUND: The sparsity of established tools for the grading of limbal stem cell deficiency hinder objective assessments of the clinical outcome of cultivated limbal epithelial cell transplantation. To advance towards the development of standards for the comparison of the outcomes of these bio-surgical protocols we have now applied a battery of recognized objective and patient-declared subjective outcome criteria to the autologous modality of cultivated limbal epithelial cell transplantation. METHODS: The prospective study involved ten patients (M/F = 9/1; mean age = 42.1 years) displaying overt unilateral limbal stem cell deficiency complying with the inclusion criteria described in Methods. Limbal biopsies were obtained from the contralateral eye and their outgrowths after 2-week cultures were transplanted on the affected eye after pannus resection. Outcomes were followed up for 12 months. The objective tests were scores for best-corrected visual acuity (BCVA); using the LogMAR scale, a multiparametric ocular surface score (OSS), and the Schirmer's test. Subjective scores were based on patient answers to a) perception of visual improvement/pain; b) the 25-item National Eye Institute Visual Function Questionnaire (NEI-VFQ 25); and c) the 12-item Ocular Surface Disease Index Questionnaire (OSDI). All procedures were performed under good manufacture practices using solely xeno-free reagents. In all cases, a single biopsy was divided into two pieces and they were expanded in order to prevent outgrowth failure. In 5 patients, both biopsies generated healthy culture sheet. In those cases the lesser outgrowth were used for immune-histological characterization. RESULTS: The experimental parallel outgrowth samples showed a similar percent of p63α+ cells. PreOp and 12-month PostOp BCVAs and OSSs were, respectively, 1.15 ± 0.70; 0.21 ± 0.13 and 7.40 ± 2.01; 2,30 ± 1.30, (p < 0.05). Patient's responses to all three question sets except ocular pain were consistent with significant improvement (p < 0.05). CONCLUSION: Objective clinical metrics demonstrate that in patients with limbal stem cell deficiency, cultivated limbal epithelial cell transplantation improves vision and ocular surface health and subjective visual perceptions.

Systemic Immunosuppression for Limbal Allograft and Allogenic Limbal Epithelial Cell Transplantation.

Bilateral limbal stem cell deficiency (LSCD) treatment requires the need to obtain allogenic limbal tissue for transplantation. Outcomes of different surgical techniques depend on multiple factors, including the underlying etiology, ocular surface, eyelid status and used surgical intervention. Some of the management options for bilateral LSCD include cadaveric, living related or living non-related conjunctival limbal allograft (CLAL), keratolimbal allograft (KLAL), allogenic cultured limbal epithelial transplantation (CLET) and allogenic simple limbal epithelial transplantation (SLET). Systemic immunosuppressive therapy plays a pivotal role in survival of transplanted tissue. The present review focuses on different systemic immunosuppression protocols for limbal allograft and allogenic limbal epithelial cell transplantation, with specific emphasis on different surgical techniques and their outcomes. We included all reports with details of different systemic immunosuppression protocols for limbal allograft and allogenic limbal epithelial cell transplantation. Oral cyclosporine A at different doses is the most commonly used immunosuppressive agent in limbal allograft and allogenic limbal epithelial cell transplantation. However, different studies using oral mycophenolate mofetil and tacrolimus also reported good results. In conclusion, systemic immunosuppression protocols for limbal allograft and allogenic limbal epithelial cell transplantation are not standardized. Further studies regarding different surgical techniques should assess outcomes and adverse effects of such protocols.

Expression of retinoic acid signaling components ADH7 and ALDH1A1 is reduced in aniridia limbal epithelial cells and a siRNA primary cell based aniridia model.

PAX6-related Aniridia is a sight-threatening disease involving progression of secondary glaucoma and aniridia related keratopathy (ARK). Change or loss of limbal epithelial progenitors causes epithelial surface defects. We analyzed the effect of PAX6 on mRNA expression changes with a two-step approach, as follows. First, we sequenced mRNA from limbal epithelial cells isolated from controls and aniridia patients. Second, we confirmed the bioinformatics and literature-based result list for a small interfering RNA (siRNA)-based primary aniridia cell model (PAX6 knockdown). With this approach, we expected that the genes directly influenced by PAX6 would be distinguishable from those affected secondarily by the ARK disease state. Therefore, epithelial cells were isolated from the limbus region of two patients with aniridia and cultured in keratinocyte serum-free medium. Normal control cells were obtained from the limbus region of corneal donors. For the siRNA-based aniridia cell model, cells were transfected with Lipofectamine and 5 nM siRNA against PAX6 or control treatment. All cells were lysed to yield DNA, RNA, and protein. Reduction of PAX6 protein was assessed by western blot. Aniridia and control Poly-A-enriched RNA libraries were subjected to next-generation sequencing. The differential analysis was a combination of quantification with RSEM and differential tests with edgeR. Gene lists were filtered by comparison to NCBI GEO datasets, annotated with DAVID, and manually annotated using a literature search. Based on the resulting filtered gene list, qPCR primers were purchased, and candidate genes (TP63, ABCG2, ADH7, ALDH1A1, PITX1, DKK1, DSG1, KRT12, KRT3, KRT13, SPINK6, SPINK7, CTSV, SERPINB1) were verified by qPCR on the siRNA-based aniridia cell model. We identified genes that might be regulated by PAX6 and showed that SPINK7 mRNA, which codes for a protease inhibitor, is downregulated in patients as well as in our primary aniridia cell model. ALDH1A1 and AHD7 mRNA levels were reduced in limbal epithelial cells of aniridia patients, and both transcripts were downregulated by PAX6 knockdown in our cell model. This siRNA-based aniridia cell model is a valuable tool for confirming identified PAX6-affected genes that might promote ARK pathogenesis. The model recapitulated expression changes for SPINK7, ADH7, and ALDH1A1 that were also observed in patient samples. These results provide evidence that PAX6 might drive corneal epithelial differentiation by direct or indirect control of retinoic acid signaling processes through ADH7 and ALDH1A1.

Conditional reprogrammed human limbal epithelial cells represent a novel in vitro cell model for drug responses.

In this study, we established human limbal epithelial cells (LECs) from normal limbal tissues by using Conditional Reprogramming (CR) technology (refer to CR-LEC cells in this study). We have successfully established CR-LEC cell strains from three human donors (3 out of 3), and normal rabbits (2 out of 2) and pig (1 out of 1) as well. CR-LEC cells sustained a continuous and stable proliferation status with a normal karyotype, normal response to DNA damage, well-defined structured spheres in matrigel 3D culture. Responses of CR-LEC cells to IFN α2b, Ganciclovir and 5-Fluorouracil were different, suggesting that these drugs had different toxicities to these cells as expected. More important, there was no significant difference of responses to drugs between early and late passages of CR-LEC cells (p>0.05), indicating CR-LEC cells can serve a stable normal human cell model for toxicity assessment. Toxicity tests with monolayer cultures of CR-LEC cells were measured by staining the F-actin and Dsg-1 expression. Toxicity of three drugs at LD50 concentration resulted in a gradually increased destruction of monolayer, which is, in accordance with the irritation grade of three drugs on human cornea epithelium. Therefore, CR-LEC cells provide a novel and reliable in vitro physiological cell model for corneal toxicity assessment.

Ion-sensitive in situ hydrogels of natamycin bilosomes for enhanced and prolonged ocular pharmacotherapy: in vitro permeability, cytotoxicity and in vivo evaluation.

Delivery of therapeutic molecules into the deeper ocular compartments is mainly hampered by short precorneal residence and limited transmembrane permeability of topically administered drugs. Hence, the current study was undertaken to fabricate the ion-sensitive in situ gels of natamycin (NT) bilosomes (NB) for efficient ocular delivery. The effect of cholesterol and sodium taurocholate proportion on the properties of the bilosomes were studied and the formulation with better physicochemical properties was optimized and utilized to derive the in situ gelling system (IG). The impact of type/composition of gelling agent on the formation and characteristics of the hydrogel was investigated. The hydrogel formed from IG with 0.3% w/v gellan gum showed optimal viscoelastic and adhesive characteristics. The ocular safety and cytocompatibility of NB and its IG was confirmed by corneal histology and in vitro cytotoxicity evaluation. A 6- to 9-fold enhancement in the transcorneal flux of NB demonstrated efficient ocular penetration of bilosomes. Moreover, the superior mean dose normalized NT levels in the ocular tissues of rabbits treated with optimized NB and IG illustrated the effectiveness of bilosomes loaded ion-sensitive in situ hydrogels as a potential platform for the improved and prolonged ocular pharmacotherapy.

Characterization of the corneal surface in limbal stem cell deficiency and after transplantation of cultured allogeneic limbal epithelial cells.

PURPOSE: The objective of this study was to characterize the changes that occur in the cornea during Limbal Stem Cell Deficiency (LSCD) and on the corneal surface after transplantation of ex vivo cultured allogeneic limbal epithelial transplantation (CALET). METHODS: Forty-one pannus were analyzed to characterize the changes found in the cornea in LSCD. Nineteen impression cytology samples, including 14 pannus and five corneal buttons, obtained during subsequent procedures from patients who had undergone CALET were examined to assess the effect of CALET and to determine the long-term fate of donor cells. The presence of donor and recipient epithelial cells in each sample was determined by short tandem repeat (STR) amplification and fluorescent-multiplex polymerase chain reaction (PCR). Phenotypic analysis of the epithelium was performed by immunohistochemistry and real-time PCR. RESULTS: The expression of lineage markers was similar between pannus and conjunctivae, but not to corneas. Objective long-term benefits from the transplantation were recorded in most cases. After CALET, the lineage markers in the excised corneal buttons and pannus showed a limbus phenotype. DNA analysis of the 19 cases showed no donor cells present on the ocular surface beyond three months after CALET. CONCLUSIONS: LSCD was characterized by ingrowth of abnormal, inflamed tissue with a conjunctival phenotype. CALET was a useful technique for restoring the ocular surface in LSCD. However, such benefits did not necessarily correlate with survival of measurable numbers of donor cells on the ocular surface. The absence of donor DNA beyond three months raises questions regarding the period of ongoing immunosuppression and the origin of the regenerated corneal epithelium.

Glutaraldehyde cross-linking of amniotic membranes affects their nanofibrous structures and limbal epithelial cell culture characteristics.

Given that the cells can sense nanometer dimensions, the chemical cross-linking-mediated alteration in fibrillar structure of collagenous tissue scaffolds is critical to determining their cell culture performances. This article explores, for the first time, the effect of nanofibrous structure of glutaraldehyde (GTA) cross-linked amniotic membrane (AM) on limbal epithelial cell (LEC) cultivation. Results of ninhydrin assays demonstrated that the amount of new cross-links formed between the collagen chains is significantly increased with increasing the cross-linking time from 1 to 24 hours. By transmission electron microscopy, the AM treated with GTA for a longer duration exhibited a greater extent of molecular aggregation, thereby leading to a considerable increase in nanofiber diameter and resistance against collagenase degradation. In vitro biocompatibility studies showed that the samples cross-linked with GTA for 24 hours are not well-tolerated by the human corneal epithelial cell cultures. When the treatment duration is less than 6 hours, the biological tissues cross-linked with GTA for a longer time may cause slight reductions in 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, and anti-inflammatory activities. Nevertheless, significant collagen molecular aggregation also enhances the stemness gene expression, indicating a high ability of these AM matrices to preserve the progenitors of LECs in vitro. It is concluded that GTA cross-linking of collagenous tissue materials may affect their nanofibrous structures and corneal epithelial stem cell culture characteristics. The AM treated with GTA for 6 hours holds promise for use as a niche for the expansion and transplantation of limbal epithelial progenitor cells.

[Ocular Surface Reconstruction with Cultivated Limbal Epithelial Cells in Limbal Stem Cell Deficiency: One-year Follow-up Results]. / Limbal Kök Hücre Yetmezliginde Kültüre Edilmis Limbal Epitel Hücreleri ile Oküler Yüzey Rekonstrüksiyonunun Bir Yillik Sonuçlari.

PURPOSE: To evaluate the 1-year follow-up results of cultivated limbal epithelial cell (CLEC) transplantation in unilateral limbal stem cell deficiency (LSCD). MATERIAL AND METHOD: One-year follow-up results of five unilateral LSCD patients who had undergone CLEC transplantation were evaluated. Parameters for this evaluation were: fluorescein staining of ocular surface, corneal vascularization and status of epithelium with slit lamp, and visual acuity. 1.5-mm limbal biopsy was performed from the superior limbus of the healthy eyes, broke into two equal pieces, expanded on human amniotic membrane (hAM) and inserts for 14 days until getting 20 mm in size. CLECs on hAMs were used directly, and cells on inserts were used after detachment procedure. The symblepharon and pannus tissues were removed, superficial keratectomy was performed. CLEC on hAMs were transplanted with the epithelial side up onto the bare corneal stroma, sutured to the conjunctiva with 10-0 nylon sutures. Free CLEC layer from insert was placed on hAM as a second layer, additional hAM was used as a protective layer all over other tissues. RESULTS: Median age was 44.4 years (14-71). The etiology was chemical burn in all patients. Median duration of symptoms was 10 years (2-18), median follow-up period was 12.6 (12-12.5) months. Preoperative best corrected visual acuities (BCVA) were light perception in three patients, counting fingers at 50 cm in one patient and 3/10 in one patient. Visions were improved in all patients. Postoperative BCVA 12 months after the surgery were between counting fingers at 3 meters to 6/10. There was a temporary hemorrhage between the two layers of hAMs in one patient at the early postoperative period. Peripheral corneal vascularization has occurred in three patients, in patient corneal vascularization has reached to the paracentral area. DISCUSSION: CLEC transplantation is an efficient treatment option for unilateral LSCD in mid-long term.

Efficient Cultivation Conditions for Human Limbal Epithelial Cells

To compare the stem niche in different culture conditions of limbal epithelial cells, the suspended human limbal epithelial cells (HLECs) were seeded on the 3T3-pretreated plates and the other suspended cells were plated on amniotic membranes (AMs) which were either cryo-preserved or freeze-dried. All were cultured for 10 to 12 days. Reverse transcription-polymerase chain reaction (RT-PCR) for ATP-binding casette, subfamily G, member 2 (ABCG2), p63, cytokeratin 12, and connexin 43 were performed in cultivated HLECs and their expression levels were compared. The mRNA expression of all markers examined showed no statistically significant differences between the cells on cryo-preserved and on freeze-dried AM. The expression of p63 and cytokeratin 12 in cultivated cells on AMs were significantly lower than those in 3T3-cocultured cells on RT-PCR and immunofluorescent staining. Cultivated HLECs on AMs showed reduced proliferation and differentiation while maintaining stem-property regardless of the preservative method of AM.

The Characteristics of Limbal Epithelial Cells Auto-Cultivated In Vivo on Amniotic Membrane in Rabbits

PURPOSE: To investigate the characteristics of the limbal epithelial cells auto-cultivated in vivo on amniotic membrane (LIVAMs) designed for the treatment of limbal stem cell deficiency. METHODS: We removed the epithelium of AM with a No.15 blade after it was blotted with 20% ethanol and made a 360 degrees stromal flap along the epithelial defect. We then mounted over-sized AM (1 mm larger in diameter than the defect) over the defect with the border of AM inserted under the flap, and performed interrupted suture with 10-0 nylon. A therapeutic contact lens was fitted over the AM and a temporary tarsorrhaphy was performed. To examine whether the limbal epithelial cells grew well onto AM, we observed the cornea after fluorescein dye staining using a slit lamp. To explore the characteristics of LIVAMs, we performed hematoxylin-eosin (H&E) staining, immunochemical staining with AK-2, AE-5, AM-3 monoclonal antibodies, and transmission electron microscopy. RESULTS: Three of four rabbits had successful epithelial growth on the amniotic membrane. The epithelial growth on the amniotic membrane was stained using immunohistochemical staining (AK-2, AE-5). Electron microscopy showed a structure similar to that of a normal corneal epithelium. CONCLUSIONS: The technique of auto-cultivation of limbal epithelial cells in vivo on amniotic membrane can be an efficient and convenient method and preserves the characteristics of limbal epithelial cells for the treatment of limbal stem cell deficiency.

Morphology and Adhesion Complex of Cultured Epithelium, on Amniotic Membrane in Vitro and in Vivo

PURPOSE: The morphologic characteristics and adhesion complex formation of cultured limbal epithelium of rabbit on amniotic membrane, in vitro and in vivo. METHODS: Rabbit limbal explants were cultured in vitro on amniotic membrane for 4 weeks. In the in vivo culture, the rabbit corneal epithelium was removed. Next, a tunnel was created at the limbus and, the edge of amniotic membrane was secured in the tunnel and cultured for 4 weeks. The proliferation of epithelium on the amniotic membrane was observed for 4 weeks at 1 week intervals. RESULTS: AE-5 immunohistochemical staining was positive and PAS staining was negative for cultured rabbit limbal epithelium, in vitro and in vivo. Hematoxylin and Eosin staining showed the morphologic characteristics of normal rabbit corneal epithelium in both groups. Transmission electron microscopy performed at an interval of 1 week showed adhesion complex by 3 weeks of in vitro culture, and no significant change was seen until week 4. The formation of the adhesion complex was shown starting at week 1 of in vivo culture and increased until week 4. CONCLUSIONS: The morphology of corneal limbal epithelium of rabbits cultured on amniotic membrane in vitro and in vivo, did not differ significantly compared with normal rabbit epithelium. In vivo culture resulted in more a normal-looking adhesion complex compared with the in vitro culture.

The Study for Limbal Epithelial Cell Movement according to the Position of Limbal Transplant in Rabbits

PURPOSE: This study aimed to evaluate the limbal epithelial cell movement according to the position of limbal transplant in rabbits. METHODS: We performed autologous conjunctival limbal transplantation in rabbits, in situ (directed corneal end toward cornea and conjunctival end toward conjunctiva) or in versus (directed corneal end toward conjunctiva and conjunctival end toward cornea) manner. We performed 360o whole autologous conjunctival limbal transplantation in quadrant fashion. Twelve months later, we observed the gross cornea under a slit lamp, and then observed corneal tissue with PAS staining using light microscopy and immunohistochemical and immunofluorescence staining using monoclonal antibody. As mentioned above, the conjunctival limbal tissue had been transplanted in situ and in versus manner for confocal microscopy. However, before transplantation, we performed organ culture of the limbal epithelial cells with CellTracker(TM) Green CMFDA dye (Molecular Probes, USA). Two weeks later, we observed the corneal and limbal epithelial cells using the confocal microscope. RESULTS: The slit lamp examinations of the cornea showed no new vessels and clear in both specimens. There were no goblet cells and the corneal epithelial arrangement was normal under light microscopy. There were positive immunohistochemical stains for cornea-specific AK2 and AE5, and negative immunofluorescence stains for goblet cell-specific AM3 patterns in both in situ and in versus specimens. CellTracker(TM) Green CMFDA dye up-taken epithelial cells were not presented in the conjunctival epithelial side, but were in the corneal epithelial side in both specimens. CONCLUSIONS: We confirmed that the epithelial cells with characteristics of corneal epithelium moved toward the corneal center at any positions of limbal transplants in rabbits.

Effect of cellular activation of ultra low temperature preserved limbal epithelial cells of rabbit cultured in vitro / 眼科

0.05).The morphological characteristics were similar and positive on HE staining and AE5 immunohistochemical staining.PAS staining were negative in two groups.Conclusion The cellu- lar biological activations of limbal epithelial cells are decreased in the ultra low temperature preservation condition,but the affect are limit and can't change the property of corneal limbal epithelial cells and application for ocular surface reconstruction.(Ophthalmol CHN,2008,17:108-112)