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Human corneal epithelial cells are continuously replenished by limbal stem cells to maintain ocular homeostasis and normal visual function.Some ocular surface injuries or diseases, such as thermochemical burns and Stevens-Johnson syndrome, can result in limbal stem cell deficiency, severely compromising patients' visual acuity.Non-limbal autologous epithelial tissue transplantation should be considered when both eyes are affected.Oral mucosal epithelial cells are an important source of seed cells for ocular surface reconstruction.What's more, cell sheets constructed ex vivo have achieved good results in clinical applications.This article reviews the research progress on the use of oral mucosal epithelial cells for the treatment of limbal stem cell deficiency, focusing on various factors including carriers, culture conditions, and techniques that may affect the culture of oral mucosal epithelial sheets.In addition, the strengths and weaknesses of the application of cell sheets in clinical ocular surface transplantation are also discussed, so as to provide a research direction for the safe and effective reconstruction of ocular surface epithelium.

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Rat limbal niche cells (LNCs) have been proven to induce transdifferentiation of oral mucosal epithelial cells (OMECs) into corneal epithelial-like cells termed transdifferentiated oral mucosal epithelial cells (T-OMECs). This investigation aimed to evaluate the effect of subconjunctival T-OMEC injections on alkali-induced limbal stem cell deficiency (LSCD) in rats. LNCs were cocultured with OMECs in the Transwell system to obtain T-OMECs, with NIH-3T3 cells serving as a control. Subconjunctival injection of single T-OMEC or OMEC suspension was performed immediately after corneal alkali injury. T-OMECs were prelabeled with the fluorescent dye CM-DiI in vitro and tracked in vivo. Corneal epithelial defect, opacity, and neovascularization were quantitatively analyzed. The degree of corneal epithelial defect (from day 1 onward), opacity (from day 5 onward), and neovascularization (from day 2 onward) was significantly less in the T-OMEC group than in the OMEC group. Cytokeratin 12 (CK12), pigment epithelium-derived factor, and soluble fms-like tyrosine kinase-1 were expressed at a higher rate following T-OMEC injection. Some CM-DiI-labeled cells were found to be coexpressed with CK12, Pax6, and ΔNp63α in the corneal epithelium after subconjunctival injection. Subconjunctival injection of T-OMECs prevents conjunctival invasion and maintains a normal corneal phenotype, which might be a novel strategy in the treatment of LSCD.

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During the transition from human oral mucosal epithelial cells (HOMEC) to oral squamous cell carcinoma cells (Cal27), the cells must have undergone a precancerous state. To explore the malignant rule of HOMEC, plv-HOMEC was used as a model cell for the precancerous state to investigate plv-HOMEC's apoptosis by comparing human oral mucosal epithelial cells established by Lentivirus-mediated hTERT (plv-HOMEC) with HOMEC and human Cal27. The lentiviral particles overexpressing hTERT were packaged and transfected into primary HOMEC to obtain plv-HOMEC. Expression levels of NF-κB were detected in the cytoplasm and nucleus of Cal27, plv-HOMEC and HOMEC. The level of intracellular reactive oxygen species was measured to verify the endoplasmic reticulum pathway, cytochrome C expression was detected to verify the mitochondrial pathway, and FasL gene expression was detected to verify the death receptor apoptosis pathway. The total expression of NF-κB in plv-HOMEC increased, mainly due to the greater nuclear import of NF-κB, but it was still much lower than Cal27. The endoplasmic reticulum apoptosis pathway of plv-HOMEC was not significantly affected, and there were no significant differences between them and the HOMEC cells; the mitochondrial apoptosis pathway of plv-HOMEC was inhibited, and the expression of Cyt C was very close to that of Cal27, indicating that the characteristics of plv-HOMEC are so familiar with cancer cells; the death receptor apoptosis pathway of plv-HOMEC was also inhibited, and in this apoptotic pathway, plv-HOMEC were more similar to cancer cells than to HOMEC cells. The present data suggest that NF-κB nucleation may increase in the early stage of healthy cells' carcinogenesis, followed by inhibition of the mitochondrial pathway and the death receptor apoptotic pathway.

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Destruction or dysfunction of limbal epithelial stem cells (LESCs) leads to unilateral or bilateral limbal stem cell deficiency (LSCD). Fifteen years have passed since the first transplantation of ex vivo cultivated oral mucosal epithelial cells (COMET) in humans in 2004, which represents the first use of a cultured non-limbal autologous cell type to treat bilateral LSCD. This review summarizes clinical outcomes from COMET studies published from 2004 to 2019 and reviews results with emphasis on the culture methods by which grafted cell sheets were prepared.

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BACKGROUND: Although estrogen deficiency has been proposed as a risk factor for oral mucosal inflammatory diseases in post-menopausal women, the mechanisms involved remain unclear. This study aimed to investigate the effect of 17ß-estradiol (E2) on the inflammatory response stimulated by interleukin-1 beta (IL-1ß) in human oral mucosal epithelial cells (hOMECs) and its possible mechanism. METHODS: Primary hOMECs were obtained from female infants and cultured in keratinocyte growth medium. The hOMECs at second passage were collected and stimulated by 10-7  mol/L ICI182,780 or 10-7  mol/L G1 for 1 hour, E2 (10-7  mol/L, 10-8  mol/L, 10-9  mol/L) for 36 hour, 100 ng/mL IL-1ß for 12 hours, respectively. Human beta-2 defensin (hBD-2), tumor necrosis factor-alpha (TNF)-α, IL-6, IL-8, estrogen receptor-alpha (ERα), estrogen receptor-beta (ERß), and G protein-coupled receptor 30 (GPR30) mRNA levels and protein levels were measured by real-time quantitative polymerase chain reaction (RT-qPCR), enzyme-linked immunosorbent assay (ELISA), and Western Blot (WB), respectively. RESULTS: Expression of hBD-2 and inflammatory cytokines increased after IL-1ß stimulation, which was down-regulated by E2 pre-treatment. With ICI182,780, the suppression of E2 on hBD-2 mRNA was attenuated. With G1, the mRNA expression and protein expression of hBD-2 were reduced. CONCLUSION: Pre-treatment of hOMECs with E2 at physiological concentrations inhibited the IL-1ß-induced expression of hBD-2 and inflammatory cytokines. The protective effects of E2 suggest its potential use treating oral inflammatory diseases in clinical practice.

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OBJECTIVE: This study aimed to investigate the effects of cigarette smoke (extract) on autophagy and apoptosis in oral mucosa epithelial cells. METHODS: The effects of cigarette smoke extract (CSE) on autophagy and apoptosis in oral epithelial cells were studied in vivo and in vitro. Leuk-1 cells were administered cigarette smoke extract or chloroquine (CQ) and rapamycin (RAPA) at different concentrations. Immunoblotting, immunofluorescence, Western blotting and flow cytometry were used to detect autophagy-related protein and apoptosis levels, screen the optimal concentration and stimulation time, and verify the effect of CSE stimulation on autophagy and apoptosis in leuk-1 cells. Meanwhile, autophagy expression in epithelial cells from the local oral tissues of mice who had smoked for 5 months was detected. RESULTS: Under CS stimulation, LC3-II and Beclin-1, the key proteins of leuk-1 autophagy, were upregulated in a concentration- and time-dependent manner. In addition, CS significantly upregulated the expression of Cleaved caspase-3 (C-casp3), a protein involved in apoptosis. However, under stimulation with CQ, autophagy in leuk-1 cells was inhibited and the level of C-casp3 and the apoptosis rate were increased. The autophagy activator RAPA significantly reduced the level of C-casp3 and apoptosis rate in leuk-1 cells. CONCLUSION: The results of this study indicate that CS can simultaneously activate autophagy and apoptosis in mouse and human oral epithelial cells, that autophagy inhibition can aggravate the CSE-triggered apoptosis of oral epithelial cells, and that autophagy induction can inhibit the CSE-triggered apoptosis of oral epithelial cells. Autophagy is suggested to play a protective role in the CSE-induced apoptosis of oral epithelial cells. Further studies are needed to explore the concrete mechanisms underlying the regulatory effects of CS-induced apoptosis and to gain in-depth insight into the complex interactions between apoptosis and autophagy.

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BACKGROUND: Candida albicans (C albicans) is the most common fungal pathogen causing opportunistic infections. IL17 (IL17A) is a vital mediator of antifungal immunity. The aim of the study was to investigate the effect of recombinant human interleukin 17A (rhIL17A) on human oral mucosal epithelial cells (hOMECs) defending against C albicans infection. METHODS: Human oral mucosal epithelial cells were divided into four groups: C albicans+ (MOI = 0.1), rhIL17A+ (100 µg/L), rhIL17A + C albicans+ (MOI = 0.1, rhIL17A:100 µg/L) and blank control. Then, C albicans growth was observed after 24 hours. Human beta-2 defensin (hBD-2), S100A8 and LL-37 in supernatants and their mRNAs in cells were measured by enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction, respectively. RESULTS: In C albicans+ group, C albicans hyphae formation and the death of infected hOMECs were observed. However, in the rhIL17A + C albicans+ group, IL17 inhibited both hypha formation, and C albicans from infecting hOMECs and its further growth. There was no statistical significance in adhesion rates of C albicans to hOMECs. Compared with the control group, the level of hBD-2 mRNA has increased, while hBD-2 and hBD-2 mRNA levels in the rhIL17A + C albicans+ group were the highest. Both hBD-2 and hBD-2 mRNA levels were higher in the rhIL17A+ group than in the C albicans+ group. S100A8 and LL-37 mRNAs have similar trend, and both upregulated after treatment with rhIL17A; however, protein levels were undetectable. CONCLUSION: Recombinant human interleukin 17A may inhibit C albicans from infecting hOMECs by affecting the growth and reproduction of C albicans as well as the formation of hyphae. Besides, rhIL17A might induce hBD-2, S100A8 and LL-37 secretion from hOMECs to strengthen their anti-infective ability.

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Most cells for regenerative medicine are currently cultured manually. In order to promote the widespread use of regenerative medicine, it will be necessary to develop automated culture techniques so that cells can be produced in greater quantities at lower cost and with more stable quality. In the field of regenerative medicine technology, cell sheet therapy is an effective tissue engineering technique whereby cells can be grafted by attaching them to a target site. We have developed automated cell culture equipment to promote the use of this cell sheet regenerative treatment. This equipment features a fully closed culture vessel and circuit system that avoids contamination with bacteria and the like from the external environment, and it was designed to allow 10 cell sheets to be simultaneously cultured in parallel. We used this equipment to fabricate 50 sheets of human oral mucosal epithelial cells in five automated culture tests in this trial. By analyzing these sheets, we confirmed that 49 of the 50 sheets satisfied the quality standards of clinical research. To compare the characteristics of automatically fabricated cell sheets with those of manually fabricated cell sheets, we performed histological analyses using immunostaining and transmission electron microscopy. The results confirmed that cell sheets fabricated with the automated cell culture are differentiated in the same way as cultures fabricated manually.

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Hyaluronan (HA), a major component of the extracellular matrix, plays a key role in cell proliferation, growth, survival, polarization and differentiation. We investigated the optimization of a HA hydrogel scaffold for culture of human oral mucosal epithelial cells (OMECs) for potential application in limbal stem cell therapy. The effect of the optimized scaffold on OMEC cell sheet morphology, cell metabolic activity and expression of genes associated with stemness, adherence and cell damage was studied. The results indicate that HA hydrogels crosslinked with polyethylene glycol diacrylate (PEGDA) failed to support OMEC attachment and growth. However, HA hydrogel scaffolds dried for three days and coated with 1 mg/mL collagen IV produced a full OMEC sheet. Cell morphology was comparable to control after three weeks culture, maintaining 76% metabolic activity. Of apoptosis-related genes, the pro-apoptotic markers CASP3 and BAX2 were upregulated and downregulated, respectively, compared to control whereas the anti-apoptotic marker BCL2 was downregulated. The expression level of stemness genes ΔNp63α and ABCG2 was significantly higher than control. Genes associated with improved scar-less wound healing (integrin-V) and protection of the ocular surface (cadherin-1) had ~3-fold increased expression. These data suggest that our optimized HA-hydrogel scaffold could enhance culture of OMEC cell sheets for use in ocular reconstruction.

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OBJECTIVE: The aim of this study was to investigate the effects of Candida albicans on the production of defense effector molecules by human oral mucosal epithelial cells in vitro. DESIGN: Immortalized human oral mucosal epithelial (Leuk-1) cells and C. albicans strain 5314 were cocultured at different cell-to-C. albicans ratios. The viability of Leuk-1 cells was determined by MTT and RTCA measurements. The secretory levels of multiple defense effector molecules were determined by Enzyme-linked immunosorbent assay (ELISA). RESULTS: Our results indicated that C. albicans significantly decreased the secretion of IgG, cystatin C, lactoferrin, and TGF-ß1 in a dose-dependent manner and remarkably reduced the production of IgA independent of the cell-to-C. albicans ratio. However, C. albicans clearly enhanced the secretion of IgM, galectin-3, P-selectin, granzyme B and perforin. CONCLUSION: These results suggest that C. albicans may exert a regulatory role in the defense response of oral mucosal epithelial cells by altering secretory levels of defense effector molecules.

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BACKGROUND: Intrauterine adhesion (IUA) is characterized by progressive intrauterine fibrosis as a consequence of trauma to the basal layer of the endometrium. In an attempt to relieve IUA, many approaches have been applied in the clinic but show limited effects. In this study, we investigated the effect of autologous oral mucosal epithelial cells (OMECs) seeded on decellularized and lyophilized amniotic membrane (DL-AM) on preventing the development of IUA in a rat model. METHODS: IUA model was established by surgical scraping of the endometrium in the left uteri (the right uteri were kept as control) of SD rats. Wounds were randomly treated as unrepaired (IUA group), repaired with DL-AM alone (DL-AM group), and DL-AM seeded with autologous OMECs (DL-AM+OMECs group), respectively, in a total of 54 rats (n = 18 each). Uterus samples were harvested for histological and immunohistochemical evaluation after 3, 7, 14, and 28 days (n = 3 in each time point) of operations. RESULTS: After surgery, the uterine cavity was observed to be filled with extensive fibrosis in the IUA and DL-AM groups, respectively, while a lower ratio of the fibrotic area was identified in the DL-AM transplantation group. Transplantation of OMECs seeded on DL-AM significantly reduced fibrosis of IUA with recovered uterine cavity and regenerated epithelium and endometrial glands as determined by CK-18 immunostaining. OMECs transplantation resulted in extensive cellular proliferation as revealed by the Ki-67 immunofluorescent staining exhibited. Meanwhile, microvessel density was significantly increased in uteri that received OMECs transplantation, which was concomitant with elevated expression of vascular endothelial growth factor. The pregnancy test (n = 6 in each group) showed successful conception in the OMEC-transplanted uteri, but not in the IUA and DL-AM groups. CONCLUSIONS: Engineered epithelium developed from DL-AM seeded with OMECs showed great potential in preventing progression of intrauterine adhesion by improved endometrial epithelium regeneration.

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BACKGROUND: Cultivated oral mucosal epithelial cells (OMECs) are widely used in the treatment of limbal stem cell deficiency (LSCD) for their ocular reconstruction capability. As the most important component of the limbal microenvironment, limbal niche cells (LNCs) play a key role in the direction of stem cell differentiation. In this study, we investigated whether LNCs can induce the transdifferentiation of rat OMECs to corneal epithelial-like cells. METHODS: We isolated OMECs and LNCs from rats by dispase and collagenase, respectively, to establish a three-dimensional or Transwell coculturing system. NIH-3T3 cells and renewed LNCs were also used as feeder layers in the Transwell system to compare their ability to support the OMECs. The airlift method was used for the culture of OMECs to obtain a stratified epithelial sheet. Cocultured OMECs were characterized by reverse-transcription polymerase chain reaction, Western blotting, hematoxylin and eosin staining, and immunohistochemistry. RESULTS: The cocultured OMECs showed corneal epithelial-like morphology and expressed the corneal epithelial markers CK12 and Pax6 in most cocultured systems. Furthermore, we found that the expression level of CK12, Pax6, and proliferation marker Ki67 was upregulated when compared with that of other groups by renewing the LNCs in the Transwell system (p < 0.05, n = 3), suggesting that this might be a potential method for improving the efficiency of transdifferentiation. The obtained stratified epithelial sheet expressed CK3 and CK12. CONCLUSION: Through coculturing OMECs and LNCs in vitro, we successfully cultivated corneal epithelial-like OMECs. This investigation is of great significance for the treatment of LSCD and ocular surface reconstruction.

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OBJECTIVE: To observe the preventive effects of oral mucosal epithelial cells (OMECs) seeded on a decellularized human amniotic membrane (dHAM) in a rat model of intrauterine adhesion (IUA). METHODS: IUAs were established by mechanical endometrial scraping in model rats. Thirty-six Sprague-Dawley rats were divided into: IUA, dHAM transplantation, and dHAM+OMECs transplantation groups (12 in each group). The oral mucosae of rats were collected, and the OMECs were cultured on the dHAM. The dHAM and dHAM+OMECs were transplanted into left scraped uteri (right uteri as controls) in the dHAM and dHAM+OMECs transplantation groups. At 3, 7, 14, and 28 days after transplantation, the uterine samples underwent histological and immunohistochemical staining. RESULTS: After endometrial scraping, the endometria and uterine cavities were obliterated. The ratios of the fibrotic areas to the whole endometrium in the IUA and dHAM transplantation group were higher than in the control group (P<0.01). In the dHAM+OMECs transplantation group, the fibrotic area significantly decreased compared to the IUA group after 7, 14, and 28 days, and the dHAM transplantation group at 14 and 28 days (P<0.01). At 14 and 28 days after surgery in the dHAM+OMECs transplantation group, the number of endometrial glands and the endometrial thickness increased (P<0.01), and regenerated epithelia were observed. Immunohistochemical assays for cytokeratin 18 demonstrated that the newly-regenerated epithelium was endometrium. CONCLUSION: OMECs can enhance repair of damaged endometrium, and promise to be novel and effective in preventing IUAs. However, studies of OMECs in patients with IUA need further exploration.

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Limbal stem cells (LSCs),the source of corneal epithelial cells,play an important role in the ocular surface. In recent years, with the development of somatic stem cell application and tissue engineering, biomaterials and cell culture technology,progress has been made on the basic researches and clinical applications of ocular surface reconstruction with ex vivo cultured limbal epithelial and oral mucosal epithelial cell sheet transplantation. However, there are several issues, including the successful clinical outcomes for ocular surface reconstruction,and the in vivo tracking of donor stem cells,remained indefinitive. This article introduced and compared recent advancements of tissue engineering techniques ex vivo cultured autologous or allogeneic limbal,oral mucosal epithelial cells in ocular surface reconstruction,so as to provide a useful direction for the future research of ocular surface reconstruction.

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AIM: To investigate human oral mucosal fibroblasts (HOMF) and human limbal fibroblasts (HLF) as alternatives to murine 3T3 feeder fibroblasts currently used to support epithelial cell expansion for the treatment of limbal epithelial stem cell deficiency. METHODS: HLF and HOMF were compared with 3T3s for their ability to support the culture of human limbal epithelial cells and human oral mucosal epithelial cells. RESULTS: HOMF, but not HLF, were equivalent to 3T3s in terms of the number of epithelial population doublings achieved. Human limbal epithelial cells co-cultured with HOMF or 3T3s had similar expression of corneal and putative stem cell markers. CONCLUSION: HOMF are a suitable and safer feeder fibroblast alternative to 3T3s for the production of epithelial cells for clinical use.

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BACKGROUND AIMS: Autologous transplantation of ex vivo cultured cells the treatment of choice for patients with limbal stem cell deficiency. The most commonly used cell sources for transplantation limbal, conjunctival or oral mucosal tissue. Protocols vary for culturing each tissue type, and there are no comparative studies on transplantation outcomes using these different culture techniques. To overcome this limitation, we devised a simple protocol that can uniformly promote growth and differentiation of cells from a limbal, conjunctival or oral mucosal biopsy into the corneal lineage. METHODS: Biopsies were cultured as explants on de-epithelialized human amniotic membrane in the presence of recombinant epidermal growth factor and insulin. Cultured cells were characterized using immunohistochemistry and quantitative reverse transcriptase polymerase chain reaction for stem/progenitor markers (ABCG2 and P63α) and differentiation markers (CK3, CK12, CK4, CK13, CK15 and CONNEXIN 43). Fluorescence-activated cell sorter analysis was performed for ABCG2. RESULTS: The results revealed that cells of all three biopsies differentiated into the corneal lineage. Positivity of CK3/12, CK4, CK12 and CONNEXIN 43 immunostaining and the relative mRNA expression of CK3, CK4, CK12, CK13, CK15 and CONNEXIN 43 could be detected in the cultured biopsies. CONCLUSIONS: Unlike tissue-specific protocols, our protocol can unequivocally promote differentiation of cells from a limbal, conjunctival or oral mucosal biopsy into the corneal lineage. This simple standardized protocol can be adapted for ocular surface reconstruction using stem cell transplantation.

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Smoking is a well-known risk factor for many systemic diseases and oral disorders. Smoking has been recognized to cause diminished defense, persistent inflammation and result in disease development. Nucleotide binding oligomerization domain 1 (NOD1) signal pathway plays a key role in innate immune and tissue homeostasis. Our recent studies confirmed that cigarette smoke extract (CSE) could inhibit NOD1 expression and affect expression levels of crucial molecules of NOD1 signaling in oral mucosal epithelial cells. In the present study, immortalized human oral mucosal epithelial (Leuk-1) cells were treated with CSE, iE-DAP (NOD1 agonist), CSE + iE-DAP, respectively. Western blotting analysis demonstrated that iE-DAP triggered NOD1 expression of leuk-1 cells in a dose-dependent manner. iE-DAP also reversed the suppressive effect of CSE on NOD1 expression and prevented the overactivation of RIP2 and P-NF-κB following CSE exposure. Real-time PCR and ELISA results confirmed that iE-DAP reversed CSE-mediated effects on the mRNA levels and releases of IL-6, IL-8, TNF-α and IFN-γ by Leuk-1 cells. Taken together, our results indicated that NOD1 activation with iE-DAP could reverse CSE-mediated effects on NOD1 signaling in human oral mucosal epithelial cells.

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A number of diseases and external factors can deplete limbal stem cells, causing pain and visual loss. Ten years have passed since the first transplantation of cultured oral mucosal epithelial cells in humans, representing the first autologous cell-based therapy for severe bilateral limbal stem cell deficiency. Its steady increase in popularity since then can be attributed to the accumulating evidence of its efficacy in reverting limbal stem cell deficiency. In this review, the focus is on clinical, and to a lesser degree laboratory, features of cultured oral mucosal epithelial transplants over the past 10 years. Comparisons with other available technologies are made. Avenues for research to stimulate further improvements in clinical results and allow worldwide distribution of limbal stem cell therapy based on oral mucosal cells are discussed. These include storage and transportation of cultured oral mucosal epithelial sheets and in vivo culture of oral mucosal epithelial cells.

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AIM OF THE STUDY: To develop a clinical grade fibrin gel for the culture of oral mucosal epithelial cells (OMEC) intended for ocular surface reconstruction in the treatment of limbal stem cell deficiency (LSCD). MATERIALS AND METHODS: Transparent fibrin gels composed of fibrinogen and thrombin were developed for the culture of epithelial cells. Oral mucosa was harvested from the buccal region of healthy volunteers and cultured as explants on fibrin gels. Tranexamic acid (TA), a clinically approved anti-fibrinolytic agent was added to prevent the fibrin gel from digesting due to cellular activity. The gels were stained for p63α (as a marker of poorly differentiated epithelial cells), CK19, CK13 and CK3 (expressed by OMEC). Epithelial cell stratification was observed using hematoxylin-eosin staining. RESULTS: Addition of TA prevented gels from dissolving during the culture period. OMEC proliferated on the fibrin gel and attained confluence over a 2-week period (±2 d) and exhibited a typical epithelial, cobblestone morphology. Basal OMEC exhibited positive staining for p63α while the superficial cells exhibited positive staining for CK3. The cells expressed a strong immunoreactivity for CK19 and CK13 suggesting that they retained a normal oral epithelial phenotype. CONCLUSION: Fibrin gels, maintained in the presence of TA, to control the rate of substrate degradation, provide a more robust yet transparent substrate for the culture and transplantation of cultured OMEC. The fibrin gels are easily standardized, the components commercially available, and produced from clinically approved materials. The resulting stratified OMEC-derived epithelium displays characteristics similar to that of a human cornea, e.g. CK3 expression. The conventional dependence on a murine feeder layer for support of epithelial cells is unnecessary with this technique and hence, provides for an attractive alternative for treatment of LSCD.

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